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US20020022257A1 - Three-dimensional structure and crystallization method of ribosome recycling factor (RRF) - Google Patents

Three-dimensional structure and crystallization method of ribosome recycling factor (RRF) Download PDF

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US20020022257A1
US20020022257A1 US09/731,487 US73148700A US2002022257A1 US 20020022257 A1 US20020022257 A1 US 20020022257A1 US 73148700 A US73148700 A US 73148700A US 2002022257 A1 US2002022257 A1 US 2002022257A1
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atom
rrf
hoh
arg
compound
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Se Suh
Kyeong Kim
Kyeong Min
Sung Ha
Soo Eom
Wook Kim
Jung Yun
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Promeditech Inc
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Assigned to SUH, SE WON, KIM, KYEONG KYU reassignment SUH, SE WON INVALID RECORDING SEE: REEL 011793, FRAME 0528. RE-RECORD TO CORRECT SERIAL NUMBER ASSIGNED BY THE PTO. Assignors: KIM, WOOK HYUN, YUN, JUNG MIN, EOM, SOO HYUN, HA, SUNG CHUL, MIN, KYEONG SIK
Assigned to PROMEDITECH, INC. reassignment PROMEDITECH, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SUH, SE WON, KIM, KYEONG KYU
Publication of US20020022257A1 publication Critical patent/US20020022257A1/en
Priority to US10/357,884 priority Critical patent/US20030199070A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the present invention relates to a crystal of ribosome recycling factor (RRF) and a method of crystallization. More particularly, the present invention relates to an RRF crystal formed by binding with a detergent and to the method of crystallization using the detergent. Further, the present invention relates to a protein complex comprising RRF or an RRF homolog and a molecule or material binding to the hydrophobic cleft formed between the two domains of RRF. The present invention further relates to a polypeptide comprising the structure of one domain of RRF which binds to the ribosome. The present invention further relates to a method for screening an RRF inhibitor using the three-dimensional structure of RRF. The present invention further relates to an RRF inhibitor comprising alkyl- ⁇ -D-maltopyranoside or its derivatives.
  • RRF ribosome recycling factor
  • Protein biosynthesis consists of three steps: initiation, elongation and termination.
  • the protein-releasing factor RF1 and RF2 in prokaryotes and eRF1 in eukaryotes
  • RF3 releasing factor 3
  • Ribosome recycling factor plays a necessary role in termination by catalyzing the hydrolysis of GTP and the release of the mRNA and tRNA from the ribosome.
  • elongation factor G elongation factor G
  • RF3 release factor 3
  • RRF has been found to directly dissociate peptidyl-tRNA as well as deacylated tRNA from the ribosome (Heurgue-Hamard et al., 1998; Grentzmann et al., 1998).
  • RRF has also been found to play a role in the prevention of errors generated in the elongation step of protein synthesis (Janosi et al., 1996).
  • RRF moves to the P-site after binding to the A-site of the ribosome (Nakamura et al., 1996) and deacylated tRNA is moved to the E-site (Janosi et al., 1996; Pavolv et al., 1997a).
  • the complex is dissociated into mRNA, tRNA and the ribosome (Kaji et al., 1998) or it may dissociate into two units: 1) the tRNA and 2) the ribosome bound to the mRNA (Pavolv et al., 1997a).
  • RRF, EF-G and GTP only catalyze the dissociation of the 50S subunit from the termination complex, and tRNA is removed from the 30S:mRNA:tRNA complex by an initiation factor, IF3(Karimi et al., 1999).
  • RRFs Genes encoding RRFs are widely distributed in prokaryotes and are found to have very high homologies (Janosi et al., 1996). As a result of sequence analysis, it was found that the genes encoding RRFs also exist in eukaryotes. However, in eukaryotes they are mostly present in organelles (Janosi et al., 1996). The RRF homolog in yeast participates in protein synthesis in mitochondria (Janai et al., 1998), but is not essential for cell growth (Kaji et al., 1998). An RRF homolog located in the chloroplast of spinach exerts an inhibitory action on a bacterial temperature-sensitive mutant (Rolland et al., 1999).
  • eukaryotic RRF is not important for cell viability and that the inhibition of the RRFs located in the organelles does not influence the synthesis of cytoplasmic proteins. This fact is also evidenced by the fact that RRF does not exist in Methanococcus jannashchii , an ancient microbe having a translation system similar to a eukaryote. Because of this, RRF is thought to be an excellent target for developing new antibacterial drugs (Kaji et al., 1998), since it is essential for prokaryotic cell viability, but not essential in eukaryotes.
  • the RRF crystal is the starting point for analyzing the three-dimensional structure of RRF, which is key to the design, screening and identification of RRF inhibitors.
  • RRF inhibitor candidates can be screened easily, and RRF inhibitors with optimum activity can be designed and identified.
  • a polypeptide may inhibit the activity of RRF by competitively binding to the RRF binding site of the ribosome, thereby preventing RRF from binding to the ribosome.
  • a crystal and a method for crystallization of RRF or an RRF homolog there is provided a crystal and a method for crystallization of RRF or an RRF homolog.
  • the RRF crystal or an RRF homolog according to the present invention can be obtained by crystallizing it with a material or compound or molecule which binds to a specific site of the RRF.
  • the RRF crystal according to the present invention includes RRF and the material which binds to the specific site of the RRF.
  • the specific site of RRF has been found to be a hydrophobic cleft formed between two domains in the three-dimensional structure of RRF identified in the present invention.
  • the two domains are formed with an L-shape and the hydrophobic cleft is formed between the two domains.
  • One domain of RRF can bind to the ribosome and the activity of RRF may be exerted within the hydrophobic cleft.
  • the inhibitors may include maltopyranosides or their derivatives identified as having RRF inhibition according to the present invention.
  • a polypeptide comprising a part or all of the coiled coil domain of RRF.
  • the polypeptide can inhibit the activity of RRF by competitively binding to the RRF binding site of the ribosome, thereby preventing RRF from binding to the ribosome.
  • FIG. 1 is a ribbon diagram of the three-dimensional structure of RRF analyzed with the RRF crystal according to the present invention.
  • FIG. 2 provides a stick model showing a detergent molecule located in a cleft of RRF drawn from a surface model of the three-dimensional structure of RRF analyzed with the RRF crystal according to the present invention.
  • FIG. 3 shows the multiple sequence alignment of RRFs derived from prokaryotes.
  • the E. coli sequence is SEQ ID NO:1
  • the H. influenza sequence is SEQ ID NO:2
  • the P. aeruginosa sequence is SEQ ID NO:3
  • the B. subtilis sequence is SEQ ID NO:4.
  • FIG. 4 shows relative movement between the two domains of the RRF in the three-dimensional structure of RRF analyzed with the RRF crystal according to the present invention.
  • FIG. 5 provides the three-dimensional structural surface models of RRF with molecules binding to the A-site of the ribosome.
  • the molecules shown are tRNA and EF-G and also shown is the structural similarities therebetween.
  • RRF ribosome recycling factor
  • Amino acids Amino acids One Letter Symbol Three Letter Symbol Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val
  • a Crystal refers to a protein of crystalline form. A crystal is to be piled up with several unit cells.
  • the word Complex refers to the form of protein when the protein is bound with one or more materials, molecules or compounds.
  • a Unit cell is the simplest and smallest volume of unit in a crystal. It is defined with a, b, c, alpha, beta and gamma (Blundell et al., 1976).
  • a Space group is an arrangement of symmetric elements in a crystal.
  • English capital letters refer to the lattice type and other numbers refer to symmetric operators.
  • An Asymmetric Unit refers to the largest unit that does not have a symmetric element in the unit cell, but is superimposed with the same shape by the symmetric operation of a space group.
  • Multiple isomorphous replacement is a method for obtaining information about the phase for three-dimensional structures by using heavy metal derivatives made from infiltrating the heavy metal solution into a crystal (Blundell et al., 1976).
  • a Molecular replacement is a method for obtaining a preliminary structure from a crystal of unknown structure by changing the locations of molecules having known structural coordinates.
  • RRF homologs referred to in this application are meant to have at least 30% amino acid similarity to RRF and to include RRF mutants.
  • One object of the present invention is to obtain an RRF crystal from a solution containing a precipitant such as polyethylene glycol with a detergent having a hydrophobic part such as decyl- ⁇ -D-maltopyranoside. It is hypothesized that the RRF crystal may not be obtained free of the detergent because it has a hydrophobic part.
  • the three-dimensional structure of RRF consists of two domains, which move relative to each other, and thus it is difficult to crystallize RRF without a means for fixing the two domains.
  • Prior to this invention other research groups had tried to crystallize the isolated RRF from E. coli with no success.
  • Various methods were used to crystallize the isolated RRF from E.
  • the RRF crystal obtained by the method described above includes a detergent having a hydrophobic part, decyl- ⁇ -D-maltopyranoside.
  • the RRF crystal of the present invention may be analyzed by X-ray diffraction to identify the three-dimensional structure of RRF.
  • FIG. 1 provides a ribbon diagram of the three-dimensional structure of RRF analyzed using the RRF crystal of the present invention.
  • the two domains are linked with linkers, the arrows indicate strands and the cylinders indicate helices.
  • the ends of amino acid and carboxylic acid are indicated respectively, and the detergent is depicted using a space filling model.
  • RRF is an open L-shaped molecule comprising two domains.
  • the N-terminal helix H 1 of the first domain is linked with the second domain.
  • Two antiparallel C-terminal ⁇ -helices H 3 and H 4 connected by a short U-turn are extended from the second domain and folded back onto the N-terminal helix H1.
  • the first domain (coil domain) consists of a three-stranded antiparallel coiled-coil about 65 ⁇ long, in which the seven-turn helix H 1 packs into the parallel eleven-turn helix H 3 and the antiparallel ten-turn helix H4.
  • the two-turn overhang helices on the C-terminal end of H 3 and the N-terminal end of H 4 near the U-turn form a short two-stranded coiled-coil.
  • the second domain ( ⁇ / ⁇ domain) with approximate dimensions of 23 ⁇ 26 ⁇ 36 ⁇ has an ⁇ / ⁇ topology containing a short ⁇ -hairpin (S1 and S2) and a single a-helix (H2) packed against one face of an antiparallel four-stranded ⁇ -sheet (S3-S6).
  • Two domains are connected by two hinge residues (Arg31 and Pro103) with an approximate angle of 1100 between the lines along helix H 3 and strand S6.
  • R merge the degree of accordance between measurements of diffraction intensities when they are measured repeatedly.
  • R factor the difference between the diffraction intensities obtained by experiment and calculated in the model.
  • R free R factor calculated by using diffraction points excluded during the optimum process in order to measure the usefulness of the obtained model.
  • FIG. 2 provides a stick model showing a detergent located in a cleft of RRF drawn by a surface model of the RRF crystal. Amino acid residues are depicted which are involved in hydrophobic bonding with their names and location numbers (Thr-106, Arg-31, Pro-103, Leu-36, Leu-37, Ile-40, Leu-87, Leu-89, Leu-102) in the drawing. The head part of the detergent is not depicted, and is not involved in the hydrophobic bonding.
  • FIG. 3 shows multiple sequence alignments of RRFs derived from prokaryotes.
  • the sequences are from the following prokaryotes: E. coli, Haemophilus influenzae ( H. inf ), Psuedomonas aeuruginosa ( P. aeu ) and Bacillus subtillis ( B. sub ).
  • the secondary structure of the E. coli RRF is indicated by an arrow for a ⁇ -strand and by a cylinder for an ⁇ -helix.
  • the amino acid residues that make contact with the alkyl chain of the detergent are marked with asterisks (*).
  • the alkyl chain of the detergent n-decyl- ⁇ -D-pyranomaltoside is located in the hydrophobic cleft formed at the joint between the two domains.
  • Several hydrophobic amino acid residues form the hydrophobic cleft and interact hydrophobically with the alkyl chain of the detergent.
  • FIG. 3 from the comparisons of the protein sequences of several RRFs it is clear that the amino acid residues involved in binding the detergent are very well conserved. Therefore, these amino acid residues are likely involved in binding other proteins or hydrophobic materials, and this binding is likely to be coupled with the activity of RRF.
  • FIG. 4 the relative movement between the two domains of the RRF in the three-dimensional structure of RRF are analyzed using the RRF crystal according to the present invention.
  • the three different crystals of RRFs are named PT1 (left side), Native1 (center) and Native2 (right side).
  • the detergent of Native1 is depicted in FIG. 4. It is believed that the movement would be more pronounced without the detergent.
  • the relative movement of the two domains is a troublesome factor in crystallization of RRF itself, as described above.
  • RRF is likely to bind to other molecules or their parts at the hydrophobic cleft formed between the movable domains.
  • the hydrophobic cleft may act as a binding site for other molecules or their parts. This means that it is likely that the hydrophobic cleft influences the function of RRF in protein synthesis.
  • RRF is an essential protein for viability of bacteria(Janosi et al., 1994)
  • RRF is an excellent target for the development of antibacterial drugs (Kaji et al., 1998) and the hydrophobic cleft of RRF is an excellent target for this purpose.
  • the material binding to the hydrophobic cleft of RRF blocks the movement between the two domains of RRF, it likely blocks the ability of RRF to function in translation.
  • Possible inhibitors can be easily screened and designed by using the three-dimensional structure of RRF according to the present invention.
  • the material can be used as an RRF inhibitor and as an antibacterial agent.
  • the RRF crystal according to the present invention is obtained from the complex of RRF with decyl- ⁇ -D-maltopyranoside. This suggests that decyl- ⁇ -D-maltopyranoside is strongly bound to the hydrophobic cleft of RRF and inhibits its movement. Therefore, since decyl- ⁇ -D-maltopyranoside can inhibit the function of RRF, it can be used as an RRF inhibitor.
  • the carbon number in the hydrophobic alkyl group of the detergent binding to the cleft of RRF is preferably about 3 to 12.
  • the hydrophobic bonding is very weak.
  • the part of alkyl group not binding to the cleft of RRF may act to repulse the other part of RRF.
  • the detergent it is preferable for the detergent to have at least one group that can participate in non-hydrophobic bonding (hydrogen bonding, dipole-dipole bonding, charge-charge bonding and so on) with non-hydrophobic residues in the circumference of or near to the hydrophobic cleft of RRF.
  • the hydrophobic alkyl group can play a role in binding to the cleft of RRF, and the non-hydrophobic group can provide the specific bonding with the non-hydrophobic amino acid residues in the circumference of or near to the cleft of RRF.
  • the detergent can maintain high affinity binding with RRF.
  • the structure of a detergent for inhibiting the function of RRF includes an alkyl group with 3-12 carbon atoms in a straight chain and a non-hydrophobic group.
  • the alkyl group of the detergent used in the present invention is preferably n-alkyl, but may be substituted with C1-C3 alkyl groups on at least one carbon of the straight chain.
  • FIG. 5 provides the three-dimensional structural surface models of RRF and other molecules binding to the A-site of the ribosome (such as tRNA and EF-G). This shows the structural similarities therebetween.
  • the model on the left is RRF
  • the center model is tRNA
  • the right model is the 3,4,and 5 domains of EF-G.
  • the white color indicates a hydrophobic surface.
  • the red and blue colors indicate negatively and positively charged surfaces, respectively.
  • the structure of RRF is similar to the structures of tRNA and EF-G, which bind to the A-site of the ribosome.
  • the domain corresponding to the coiled coil of RRF has high structural similarity to the anticodon stem of tRNA and the domain IV of EF-G. From these and other results from several experiments, it is believed likely that the site directly binding to the A-site of the ribosome is on the coiled coil domain of RRF.
  • the specific site binding to the A-site of the ribosome on the coiled coil domain can be identified using the three-dimensional structure of RRF according to the present invention and a material which binds to this specific site can be identified and designed.
  • Such molecules may prevent RRF from binding to the A-site of the ribosome and can be used as an RRF inhibitor.
  • the present invention relates to a method for identifying and designing complementary molecules which bind to the hydrophobic cleft of RRF by using the three-dimensional structure, and synthesizing the molecule to use as an antibiotic.
  • the complementary molecule can be predicted to bind to the cleft of RRF physically and structurally.
  • the binding is not likely to be covalent, but can include hydrogen bonds, ionic bonds, van der Waals and hydrophobic interactions.
  • the processes for identifying and designing RRF inhibitors are exemplified below.
  • the first step is to retrieve from a database, a molecule or its part which is predicted to bind to the hydrophobic cleft of RRF in the three-dimensional structure.
  • a molecule can be designed which, based on its three-dimensional structure, will bind to the cleft.
  • the binding is not likely to be covalent, but may include hydrogen bonds, ion bonds, van der Waals and hydrophobic interactions.
  • the compound or its part may also have a special conformation which allows it to bind the cleft. The conformation is not considered in the compound itself, but in the assumption of the compound binding with the cleft.
  • Computer modeling can perform the selection and design of the compound or fragment.
  • the computer modeling technique can analyze or calculate the affinity between the compound or fragment and the cleft. If the computer modeling determines that the material or its part selected from the database or designed, does not have sufficient affinity, it is less likely to have RRF inhibitory ability and will not be used subsequently.
  • the next processes include molecule remodeling for optimizing the affinity of the molecule to the cleft. This includes modifying the structure of the compound and exchanging substituents, preparing the remodeled compound and measuring the activity of the prepared compound. In this way, the efficiency of the compound as an RRF inhibitor may be increased, because it will exclude compounds which are not likely to have inhibitor activity.
  • the compound If computer modeling determines that the compound has sufficient affinity for the cleft of RRF, it will be prepared and the affinity will be measured in vitro as will its activity as an inhibitor. At this point, if the dissociation constant of the complex of the compound and RRF is 100 micromoles or less, the compound may be considered as a good inhibitor candidate. When the dissociation constant is 100 micromoles, 99% of the inhibitor exists in the complex and 1% of the remainder is dissociated from the complex. The less the dissociation constant is, the more the affinity of the compound to RRF is. In paragraph b below, the design of a stronger inhibitor based on the inhibitor candidate is described.
  • the process for calculating the affinity of the compound for RRF by the use of a computer modeling technique is to bind the compound to the hydrophobic cleft of RRF by rotating it along several planes and to fit it to the cleft by visual inspection on the computer screen.
  • This work may be performed with modeling programs such as Insight II, Sybyl, O, Chain and Frodo.
  • the method for inserting the compound into the hydrophobic cleft of RRF may also be performed by docking, which may be performed using modeling programs such as Quanta, Autodock, Dock, Insight II, and Sybyl.
  • the position of the compound in the binding site may be made to be more accurate by using energy minimization or molecular dynamics with standard molecular mechanics force fields such as CHARMM and AMBER.
  • Specialized computer programs may also perform the process of selecting the compound. These include:
  • MCSS (Miranker, A. and M. Karplus, “ Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method” Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)). MCSS is available from Molecular Simulations, Burlington, Mass.
  • AUTODOCK (Goodsell, D. S. and A. J. Olsen, “ Automated Docking of Substates to Proteins by Simulated Annealing”, Proteins: Structure, Function and Genetics, 8, pp. 195-202 (1990)).
  • AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.
  • DOCK (Kuntz, I. D. et al., “ A Geometric Approach to Macromolecule - Ligand Interactions”, J. Mol. Biol., 161, pp. 269-288 (1982). DOCK is available from University of California, San Francisco, Calif.
  • CAVEAT Bartlett, P. A. et al, “ CAVEAT: A Program to Facilitate the Structure - Derived Design of Biologically Active Molecules” In “Molecular Recognition in Chemical and Biological Problems”, Special Pub., Royal Chem. Soc., 78, pp. 182-196 (1989). CAVEAT is available from the University of California, Berkeley, Calif.
  • 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.). This area is reviewed in Martin, Y. C., “3 D Database Searching in Drug Design”, J. Med. Chem., 35, pp. 2145-2154 (1992)).
  • the affinity of the material to RRF may be calculated.
  • the inhibitor For the inhibitor to be effective, it should have a low energy difference between that energy calculated before and after binding.
  • the most efficient inhibitor should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole.
  • the affinity of the inhibitor may also be measured by calculating the dissociation constant of the complex of RRF and the inhibitor.
  • the dissociation constant is preferably 100 micromoles or less.
  • the inhibitor preferably also maintains the bonding with RRF stably after binding. In order to do this, electrostatic repulsion such as charge-charge interactions, dipole-dipole and charge-dipole interactions between the inhibitor and RRF should not occur.
  • the sum of electrostatic interaction should be neutral or give a positive effect to the enthalpy of the bonding.
  • Examples of programs designed for calculating such affinity are as follows: Gaussian 92, revision C [M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa. ⁇ 1992]; AMBER, version 4.0 [P. A. Kollman, University of California at San Fransisco, ⁇ 1994]; QUANTA/CHARMM [Molecular Simulations, Inc., Burlington, Mass. ⁇ 1994]; and Insight II/Discover (Biosysm Technologies Inc., San Diego, Calif., ⁇ 1994).
  • a stronger inhibitor can be made or designed. This process will be described below.
  • the compound (lead compound) selected or designed as above can be changed or modified. Atoms, substituents or a part of the structure may be altered to increase the binding affinity to RRF. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided.
  • the substituted chemical compounds may then be analyzed for fit with RRF by the same computer methods described in detail, above.
  • the present invention has identified a detergent, decyl- ⁇ -D-maltopyranoside, which was used for producing an RRF crystal by binding to the hydrophobic cleft of RRF. Therefore, decyl- ⁇ -D-maltopyranoside can be used as a lead compound for the design of RRF inhibitors by performing the same computer methods described above. If needed, an appropriate substituent may be introduced into the hydrophobic chain or alkyl chain of decyl- ⁇ -D-maltopyranoside. Alternatively, the maltose group may be changed to other appropriate groups.
  • the three-dimensional structure of the complex may be determined at high resolution (over 0.28 nm) using X-ray crystallographic methods.
  • the information gained about the interaction between RRF and the inhibitor obtained from this study may be used to modify the inhibitor and to increase the affinity of the inhibitor to RRF.
  • the inhibitor candidate for RRF optimized by this method may be selected or designed.
  • the actual inhibitory activity of the candidate toward RRF may be tested in vitro and in vivo.
  • the inhibitor candidate may then be used as an inhibitor.
  • it may be used for developing a more optimal inhibitor based on its structure. For example, by improving properties such as its affinity to RRF and its solubility using the computer modeling method, the inhibitory activity may be optimized.
  • the compound may be used as a lead compound for computer remodeling to increase the properties of RRF inhibition, solubility of the compound and so on.
  • the dissociation constant of the complex of the inhibitor and RRF may be measured experimentally in order to confirm the affinity calculated by the computer modeling method.
  • the locations of amino acid residues in the surface of the coiled coil domain are as follows: 1, 3, 4, 6, 7, 8, 10, 11, 12, 14, 15, 17, 18, 19, 107, 108, 109, 111, 112, 114, 115, 116, 118, 119, 120, 122, 123, 125, 126, 127, 129, 130, 132, 133, 134, 136, 137, 138, 140, 141, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 156, 158, 159, 161, 162, 164, 165, 166, 167, 169, 170, 172, 173, 174, 176, 177, 179, 180, and 181.
  • the amino acid residues are identified in the three-dimensional structure of RRF in FIG. 1 and the coordinates of the atoms in the structure of RRF are presented in table 2.
  • RRF can be mutated by exchanging one or more of the amino acid residues on the coiled coil domain. If an E. coli possessing the mutated RRF does not grow well, grows slowly or not at all, the mutation has very likely affected the function of RRF. Specifically, the mutation has affected the ability of RRF to bind to the ribosome due to the substitution of the amino acid residue, and thus RRF cannot function in translation. This method can be used to identify amino acid residues in the original RRF which are important in the binding of RRF to the ribosome, more specifically to the A-site of the ribosome.
  • the structure of the binding site can be identified based on the three-dimensional structure of RRF. Based on the structure of the binding site, a compound such as a peptide or other compound can be screened and designed which will fit into the three-dimensional model of the binding site. For example, If a mutant which has the following residues substituted: Met-1, Ser-3 and Asp-4 has a very low activity, then these three residues are likely to be involved in the binding of RRF to the ribosome. A compound which is complementary to the structure of the binding site of RRF but includes these three residues would prevent RRF from binding to the ribosome. The complementary compound could be identified or designed from the structure of the binding site of RRF including the three residues.
  • the computer modeling method as described above can perform the identification and design of the complementary compounds. It can also be used to remodel the compound to improve the affinity or solubility, and produce an optimized compound.
  • the optimized compound may then be prepared and the inhibitory activity for RRF can be tested in vitro and in vivo. If the test confirms that the material does indeed inhibit RRF, then the material or a derivative can be used as an antibiotic. Using the method as described above, the compound identified to have inhibitory activity may be used as a lead compound to obtain an improved inhibitor.
  • the dissociation constant of the complex may be experimentally measured.
  • the three-dimensional structure of RRF or one of its domains can be used to identify other crystal forms of RRF, the crystal structures of RRF homologs (those compounds having 30% amino acid similarity or more to RRF), the crystal structures of mutants of RRF or the crystal structure of a complex of RRF with another compound. Molecular replacement can be used for this.
  • the coordinates of E. coli RRF (table 2) should be used. This method can quickly identify the unknown crystal structure.
  • the programs used in the method are as follows:
  • CCP4 program package (Collaborative Computational Project Number 4 (1994), The CCP4 suite: programs for protein crystallography, Acta crystallogr., D 50, 760-763); It includes molecular replacement program of Amore, Polarrfn et al.
  • EPMR Korean, C. R., Gehlhaar, D. K. and Fogel, D. B. (1999), Rapid automated molecular replacement by evolutionary search, Acta Crystallogr., D 55, 484-491).
  • a polypeptide which mimics a part or all of the coiled coil domain of RRF would also bind to the A-site of the ribosome.
  • This polypeptide will compete with RRF for binding to the A-site—making it a competitive inhibitor. As a result, it would prevent RRF from binding to the ribosome.
  • the preparation of a polypeptide which includes the entire coiled coil domain of RRF may be performed using general biological techniques or chemical synthesis based on the secondary structure and the three-dimensional structure of the amino acid sequence.
  • a peptide which includes the entire coiled coil domain of RRF can be used as an inhibitor for RRF, it is not likely to be efficient due to its large size. Therefore, it will be preferable to identify an inhibitor which is an amino acid or small polypeptide which has a structure which mimics the three-dimensional coiled coil structure of the binding site of RRF to the ribosome.
  • the binding site can be identified by testing the viability of E. coli containing mutations in the coiled coil domain of RRF. Identification and design of the compound can be performed by computer modeling as described above.
  • each of the peptides might be used as an inhibitor for RRF.
  • a compound formed by appropriately linking the two peptides together might also be used as an inhibitor for RRF. The latter compound would be likely to have a higher affinity to RRF than the former peptides.
  • An alternative method for developing a compound such as an amino acid or polypeptide which has the identified structure of the binding domain is to identify the compound as above, and design or synthesize it. At this point, it would not be known if this compound will bind. The inhibitory activity of this compound against RRF will then be tested in vitro or in vivo. If the compound does have inhibitory activity against RRF, it can then be used as an inhibitor. This, then is another way of identifying the structure of the binding site in the coiled coil domain. Furthermore, when two or more compounds are identified which are located close to each other on the surface of the coiled coil domain, a new compound may be designed or synthesized by linking the two compounds. The new compound may then be tested as an inhibitor of RRF. A compound composed of two or more of these compounds will have a stronger inhibitory activity than each separate compound, because it can bind more tightly to the ribosome.
  • E. coli RRF gene was amplified by PCR using E. coli genomic DNA as a template.
  • the PCR product was restricted and ligated into an NdeI/XhoI-restricted pET22b vector (Novagen).
  • E. coli cells B834(DE3) were transformed with the pET22b-RRF plasmid and grown at 310° K in LB medium containing 50 ⁇ g ml ⁇ 1 ampicillin.
  • E. coli RRF was induced with 0.5 mM IPTG for 4 h.
  • the cells were harvested at 277 K by centrifugation (5000 g for 6 min) and resuspended in buffer A (20 mM Tris-HCl pH 8.0, 10 mM MgCl 2 , 1 mM PMSF). The cells were then sonicated. After a subsequent centrifugation for 30 min at 15000 ⁇ g, the supernatant was applied to a 5 ml HiTrap Q column (Pharmacia) equilibrated with buffer A. The flowthrough containing the RRF protein was dialyzed against buffer B (20 mM NaH 2 PO 4 pH 6.0, 10 mM MgCl 2 ) and loaded onto a 5 ml HiTrap SP column (Pharmacia) equilibrated with buffer B.
  • the protein identified in the flowthrough was concentrated by ultrafiltration (Amicon, YM 10) and loaded onto a Superdex 200 gel-filtration column (HiLoad 16/60, Pharmacia) equilibrated with buffer A containing 150 mM NaCl. The fractions containing RRF were pooled and concentrated for crystallization. The purity of RRF was checked by SDS gel electrophoresis.
  • a crystal of E. coli RRF was produced using polyethylene glycol as a precipitant in the presence of the detergent, decyl- ⁇ -D-maltopyranoside (Yun et al., 2000).
  • the procedures were as follows: The RRF protein was prepared at a concentration of 100 mg/ml in a buffer (20 mM Tris-HCl, pH 8.0, 10 MM MgCl 2 , 1 mM PMSF, 150 mM NaCl).
  • the crystal growth solution had the composition: 0.1 M MES-NaOH, pH 6.5, 10% PEG 350 MME, and 12% PEG 400. 2 ⁇ l of the prepared protein solution was aliquoted onto a cover glass to form a droplet of about 2-3 mm diameter.

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Abstract

A crystal of E. coli RRF complexed with a detergent is disclosed as well as the three-dimensional structure of the complex as analyzed by X-ray diffraction. The crystallization method and a method for identifying an inhibitor of RRF based on the identified three-dimensional structure is also disclosed. The RRF crystal was obtained in the presence of a detergent, and the three-dimensional structure of RRF was analyzed from this crystal. The active site forming a hydrophobic cleft and the binding site of RRF to the A-site of the ribosome was identified in the three-dimensional structure of RRF. The three-dimensional structure of RRF, and particularly, the active site and binding site can aid in the identification or modeling of an inhibitor for RRF. Because RRF is essential for viability in prokaryotes, but is not essential in eukaryotes the inhibitor can be used as an antibiotic.

Description

    FIELD OF THE INVENTION
  • The present invention relates to a crystal of ribosome recycling factor (RRF) and a method of crystallization. More particularly, the present invention relates to an RRF crystal formed by binding with a detergent and to the method of crystallization using the detergent. Further, the present invention relates to a protein complex comprising RRF or an RRF homolog and a molecule or material binding to the hydrophobic cleft formed between the two domains of RRF. The present invention further relates to a polypeptide comprising the structure of one domain of RRF which binds to the ribosome. The present invention further relates to a method for screening an RRF inhibitor using the three-dimensional structure of RRF. The present invention further relates to an RRF inhibitor comprising alkyl-β-D-maltopyranoside or its derivatives. [0001]
    • BACKGROUND OF THE INVENTION
  • After the discovery and use of penicillin as an antibiotic, many antibiotic materials were developed to control pathogens. However, the appearance of new pathogens and antibiotic-resistant pathogens has continuously required the development of new antibiotics. Antibiotics for the purpose of suppressing the metabolism of bacteria such as the synthesis of the cell wall or protein biosynthesis have been the main type developed from natural compounds or their derivatives. [0002]
  • Recently, advances in protein crystallography and computational chemistry have introduced a new method of structure-based drug design into the field of drug development. The new method typically involves finding inhibitors by fitting inhibitor candidates to the active site of target proteins based on the high resolution crystal structure and computing the binding intensities or affinities in the complexes. This approach is useful for developing new antibiotics. [0003]
  • Protein biosynthesis consists of three steps: initiation, elongation and termination. In the termination step, the protein-releasing factor (RF1 and RF2 in prokaryotes and eRF1 in eukaryotes) helps to release the synthesized protein at the stop codon, which is then released from the A-site of the ribosome by releasing factor 3 (RF3) (Freistroffer et al., 1997). At this point, other factors remaining in the termination complex must be recycled so that the protein biosynthesis can proceed continuously (Kaji et al. 1998). Ribosome recycling factor (RRF) plays a necessary role in termination by catalyzing the hydrolysis of GTP and the release of the mRNA and tRNA from the ribosome. The process is aided by elongation factor G (EF-G) or release factor 3 (RF3) (Pavlov et al., 1997a; Kaji et al., 1998; Grentzmann et al., 1998; FIG. 1). RRF has been found to directly dissociate peptidyl-tRNA as well as deacylated tRNA from the ribosome (Heurgue-Hamard et al., 1998; Grentzmann et al., 1998). RRF has also been found to play a role in the prevention of errors generated in the elongation step of protein synthesis (Janosi et al., 1996). The importance of recycling the ribosome in the process of protein synthesis is evidenced by the fact that the growth of bacteria is suppressed when the activity of RRF is inhibited (Janosi et al., 1998). The absence of RRF caused cell death in [0004] Escherichia coli (Janosi et al., 1994), which may be due to increased translation errors (Janosi et al., 1996) and unscheduled reinitiation (Ryoji et al., 1981; Janosi et al., 1998).
  • The mechanism for the prevention of errors in the translation process or in the process of recycling of the ribosome is still unknown. RRF has been found to bind to the A-site of the ribosome and its binding is competitive with the binding of releasing factor 1 (RF1) (Pavlov et al., 1997b). Therefore, it is predicted, based on the binding affinity to the ribosome, that RRF will have structural similarity to other translation factors which bind to the A-site of the ribosome. Therefore, RRF is thought to also mimic the tRNA-like structure proposed in other translation factors (Nakamura et al., 1996; Brock et al., 1998). According to one model for recycling of the ribosome, RRF moves to the P-site after binding to the A-site of the ribosome (Nakamura et al., 1996) and deacylated tRNA is moved to the E-site (Janosi et al., 1996; Pavolv et al., 1997a). At this point, the complex is dissociated into mRNA, tRNA and the ribosome (Kaji et al., 1998) or it may dissociate into two units: 1) the tRNA and 2) the ribosome bound to the mRNA (Pavolv et al., 1997a). A mechanism proposed by more recent research differs from this in that RRF, EF-G and GTP only catalyze the dissociation of the 50S subunit from the termination complex, and tRNA is removed from the 30S:mRNA:tRNA complex by an initiation factor, IF3(Karimi et al., 1999). [0005]
  • Genes encoding RRFs are widely distributed in prokaryotes and are found to have very high homologies (Janosi et al., 1996). As a result of sequence analysis, it was found that the genes encoding RRFs also exist in eukaryotes. However, in eukaryotes they are mostly present in organelles (Janosi et al., 1996). The RRF homolog in yeast participates in protein synthesis in mitochondria (Janai et al., 1998), but is not essential for cell growth (Kaji et al., 1998). An RRF homolog located in the chloroplast of spinach exerts an inhibitory action on a bacterial temperature-sensitive mutant (Rolland et al., 1999). Therefore, it may be concluded that eukaryotic RRF is not important for cell viability and that the inhibition of the RRFs located in the organelles does not influence the synthesis of cytoplasmic proteins. This fact is also evidenced by the fact that RRF does not exist in [0006] Methanococcus jannashchii, an ancient microbe having a translation system similar to a eukaryote. Because of this, RRF is thought to be an excellent target for developing new antibacterial drugs (Kaji et al., 1998), since it is essential for prokaryotic cell viability, but not essential in eukaryotes. Although there is a chance that such an inhibitor may act on the mitochondria of eukaryotes, it can still be utilized in view of the fact that widely used antibiotics such as tetracycline and erythromycin also target protein synthesis in mitochondria. Further, from the results of recent studies on the RRF of P. aeruginosa as well as E. coli, it has been found that RRF is exchangeable between the two microbes. This fact suggests that an inhibitor with broad antibiotic specificity for prokaryotes can be developed from studies on E. coli RRF.
    • SUMMARY OF THE INVENTION
  • It is, therefore, an object of the present invention to provide an RRF crystal and its crystallization method. The RRF crystal is the starting point for analyzing the three-dimensional structure of RRF, which is key to the design, screening and identification of RRF inhibitors. [0007]
  • It is another object of the present invention to provide a method for identifying RRF inhibitors by means of the three-dimensional structure of RRF. By the present invention, RRF inhibitor candidates can be screened easily, and RRF inhibitors with optimum activity can be designed and identified. [0008]
  • It is a further object of the present invention to provide RRF inhibitors including maltopyranosides and their derivatives, which are identified as inhibiting RRF. [0009]
  • It is still a further object of the present invention to provide a polypeptide having a structure comprising part or all of the coiled coil domain of RRF. Such a polypeptide may inhibit the activity of RRF by competitively binding to the RRF binding site of the ribosome, thereby preventing RRF from binding to the ribosome. [0010]
  • In accordance with one embodiment of the present invention, there is provided a crystal and a method for crystallization of RRF or an RRF homolog. The RRF crystal or an RRF homolog according to the present invention can be obtained by crystallizing it with a material or compound or molecule which binds to a specific site of the RRF. The RRF crystal according to the present invention includes RRF and the material which binds to the specific site of the RRF. The specific site of RRF has been found to be a hydrophobic cleft formed between two domains in the three-dimensional structure of RRF identified in the present invention. In the three-dimensional structure of RRF according to the present invention, the two domains are formed with an L-shape and the hydrophobic cleft is formed between the two domains. One domain of RRF can bind to the ribosome and the activity of RRF may be exerted within the hydrophobic cleft. [0011]
  • In accordance with a further embodiment of the present invention, there is provided a method for identifying RRF inhibitors based on the three-dimensional structure of RRF. The inhibitors may include maltopyranosides or their derivatives identified as having RRF inhibition according to the present invention. [0012]
  • In accordance with a further embodiment of the present invention, there is provided a polypeptide comprising a part or all of the coiled coil domain of RRF. The polypeptide can inhibit the activity of RRF by competitively binding to the RRF binding site of the ribosome, thereby preventing RRF from binding to the ribosome.[0013]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • The above and other objects and features of the present invention will become apparent from the following description of preferred embodiments given in conjunction with the accompanying drawings, in which: [0014]
  • FIG. 1 is a ribbon diagram of the three-dimensional structure of RRF analyzed with the RRF crystal according to the present invention. [0015]
  • FIG. 2 provides a stick model showing a detergent molecule located in a cleft of RRF drawn from a surface model of the three-dimensional structure of RRF analyzed with the RRF crystal according to the present invention. [0016]
  • FIG. 3 shows the multiple sequence alignment of RRFs derived from prokaryotes. The [0017] E. coli sequence is SEQ ID NO:1, the H. influenza sequence is SEQ ID NO:2, the P. aeruginosa sequence is SEQ ID NO:3, and the B. subtilis sequence is SEQ ID NO:4.
  • FIG. 4 shows relative movement between the two domains of the RRF in the three-dimensional structure of RRF analyzed with the RRF crystal according to the present invention. [0018]
  • FIG. 5 provides the three-dimensional structural surface models of RRF with molecules binding to the A-site of the ribosome. The molecules shown are tRNA and EF-G and also shown is the structural similarities therebetween.[0019]
    • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • The invention will be illustrated by the following preferred embodiments with reference to the accompanying drawings. [0020]
  • Abbreviations and Definitions [0021]
    1) Abbreviations
    RRF: ribosome recycling factor
    Amino acids
    Amino acids One Letter Symbol Three Letter Symbol
    Alanine A Ala
    Arginine R Arg
    Asparagine N Asn
    Aspartic acid D Asp
    Cysteine C Cys
    Glutamine Q Gln
    Glutamic acid E Glu
    Glycine G Gly
    Histidine H His
    Isoleucine I Ile
    Leucine L Leu
    Lysine K Lys
    Methionine M Met
    Phenylalanine F Phe
    Proline P Pro
    Serine S Ser
    Threonine T Thr
    Tryptophan W Trp
    Tyrosine Y Tyr
    Valine V Val
  • 2) Definitions [0022]
  • The definitions used in this specification are as follows: [0023]
  • A Crystal refers to a protein of crystalline form. A crystal is to be piled up with several unit cells. [0024]
  • The word Complex refers to the form of protein when the protein is bound with one or more materials, molecules or compounds. [0025]
  • A Unit cell is the simplest and smallest volume of unit in a crystal. It is defined with a, b, c, alpha, beta and gamma (Blundell et al., 1976). [0026]
  • A Space group is an arrangement of symmetric elements in a crystal. In the definition of space group, English capital letters refer to the lattice type and other numbers refer to symmetric operators. [0027]
  • An Asymmetric Unit refers to the largest unit that does not have a symmetric element in the unit cell, but is superimposed with the same shape by the symmetric operation of a space group. [0028]
  • Multiple isomorphous replacement is a method for obtaining information about the phase for three-dimensional structures by using heavy metal derivatives made from infiltrating the heavy metal solution into a crystal (Blundell et al., 1976). [0029]
  • A Molecular replacement is a method for obtaining a preliminary structure from a crystal of unknown structure by changing the locations of molecules having known structural coordinates. [0030]
  • The RRF homologs referred to in this application are meant to have at least 30% amino acid similarity to RRF and to include RRF mutants. [0031]
  • Crystallization of RRF and Structure of the Crystal [0032]
  • One object of the present invention is to obtain an RRF crystal from a solution containing a precipitant such as polyethylene glycol with a detergent having a hydrophobic part such as decyl-β-D-maltopyranoside. It is hypothesized that the RRF crystal may not be obtained free of the detergent because it has a hydrophobic part. As described below, the three-dimensional structure of RRF consists of two domains, which move relative to each other, and thus it is difficult to crystallize RRF without a means for fixing the two domains. Prior to this invention, other research groups had tried to crystallize the isolated RRF from [0033] E. coli with no success. Various methods were used to crystallize the isolated RRF from E. coli in solutions with various concentrations of the protein under various crystallization conditions with no success. The failure was likely caused due to the fact that the crystallization was not performed with a detergent having a hydrophobic part. It is likely that the detergent is necessary because it binds to the cleft formed between the two domains of RRF, thereby eliminating the relative movement of the two domains. Therefore, in order to crystallize RRF or an RRF mutant (or RRF homologue) isolated from E. coli or other bacteria, it is likely to be necessary to use a material which binds to the hydrophobic cleft of RRF as described below, preferably a detergent having a hydrophobic part.
  • Structure of RRF Crystal [0034]
  • The RRF crystal obtained by the method described above includes a detergent having a hydrophobic part, decyl-β-D-maltopyranoside. The RRF crystal of the present invention may be analyzed by X-ray diffraction to identify the three-dimensional structure of RRF. The X-ray diffraction data suggests that the RRF crystal of the present invention has a long stick shape with a unit cell of a=b=48.06 Å and c=142.27 Å, and a space group of P3[0035] 121.
  • The three-dimensional structure of RRF is depicted in FIG. 1, which provides a ribbon diagram of the three-dimensional structure of RRF analyzed using the RRF crystal of the present invention. In FIG. 1, the two domains are linked with linkers, the arrows indicate strands and the cylinders indicate helices. The ends of amino acid and carboxylic acid are indicated respectively, and the detergent is depicted using a space filling model. [0036]
  • As shown in table 1, the three-dimensional structure of RRF was determined with 0.23 nm resolution to form a three-dimensional model for 1437 of the protein atoms, 230 of the water molecules and 16 atoms of decyl-β-D-maltopyronoside. The three-dimensional coordinate of each atom in the three-dimensional structure of RRF is shown in table 2. As shown in FIG. 1, RRF is an open L-shaped molecule comprising two domains. The N-terminal helix H[0037] 1 of the first domain is linked with the second domain. Two antiparallel C-terminal α-helices H3 and H4 connected by a short U-turn are extended from the second domain and folded back onto the N-terminal helix H1. Thus, the first domain (coil domain) consists of a three-stranded antiparallel coiled-coil about 65 Å long, in which the seven-turn helix H1 packs into the parallel eleven-turn helix H3 and the antiparallel ten-turn helix H4. The two-turn overhang helices on the C-terminal end of H3 and the N-terminal end of H4 near the U-turn form a short two-stranded coiled-coil. The second domain (α/β domain) with approximate dimensions of 23×26×36 Å has an α/β topology containing a short β-hairpin (S1 and S2) and a single a-helix (H2) packed against one face of an antiparallel four-stranded β-sheet (S3-S6). Two domains are connected by two hinge residues (Arg31 and Pro103) with an approximate angle of 1100 between the lines along helix H3 and strand S6.
    TABLE 1
    X-ray diffraction data for RRF
    Data collection
    Resolution (Å) 20.0-2.3 (2.38-2.30)
    Rmerge (%) 4.8 (23.0)
    Completeness (%) 96.2 (97.7)
    No. of unique reflections 8,691 (858)
    Redundancy 3.34
    Refinement
    Resolution (Å) 20.0-0.3
    Rfactor/Rfree (%) 22.8/29.7
    No. of reflections (2 α cut off) 8,391
    No. of solvent atoms 230
    No. of protein atoms 1437
    No. of Heteroatoms 16
    Mean B factor (Å2) 53.9
    R.m.s.d. in binding length (Å) 0.010
    R.m.s.d in binding angle (°) 1.63
  • The terms in the table are as follows: [0038]
  • Resolution: the distance between two plates when the diffraction conditions of Bragg are satisfied. [0039]
  • R[0040] merge: the degree of accordance between measurements of diffraction intensities when they are measured repeatedly.
  • R[0041] factor: the difference between the diffraction intensities obtained by experiment and calculated in the model.
  • R[0042] free: Rfactor calculated by using diffraction points excluded during the optimum process in order to measure the usefulness of the obtained model.
  • Hydrophobic Cleft and Movement of Domains [0043]
  • FIG. 2 provides a stick model showing a detergent located in a cleft of RRF drawn by a surface model of the RRF crystal. Amino acid residues are depicted which are involved in hydrophobic bonding with their names and location numbers (Thr-106, Arg-31, Pro-103, Leu-36, Leu-37, Ile-40, Leu-87, Leu-89, Leu-102) in the drawing. The head part of the detergent is not depicted, and is not involved in the hydrophobic bonding. [0044]
  • FIG. 3 shows multiple sequence alignments of RRFs derived from prokaryotes. The sequences are from the following prokaryotes: [0045] E. coli, Haemophilus influenzae (H. inf), Psuedomonas aeuruginosa (P. aeu) and Bacillus subtillis (B. sub). The secondary structure of the E. coli RRF is indicated by an arrow for a β-strand and by a cylinder for an α-helix. The amino acid residues that make contact with the alkyl chain of the detergent are marked with asterisks (*). The mutated residues identified in temperature-sensitive mutants of RRF (Janosi et al., 1998) are indicated by # marks. The heptad pattern (abc defg) of each residue in the coiled-coil is also shown. In the four sequences, identical residues are boxed in black and homologous residues are boxed in gray.
  • As shown in FIG. 2, the alkyl chain of the detergent, n-decyl-β-D-pyranomaltoside is located in the hydrophobic cleft formed at the joint between the two domains. Several hydrophobic amino acid residues form the hydrophobic cleft and interact hydrophobically with the alkyl chain of the detergent. As shown in FIG. 3, from the comparisons of the protein sequences of several RRFs it is clear that the amino acid residues involved in binding the detergent are very well conserved. Therefore, these amino acid residues are likely involved in binding other proteins or hydrophobic materials, and this binding is likely to be coupled with the activity of RRF. The contact of detergent in the hydrophobic cleft will decrease the relative movement between the two domains, which are linked through the cleft. In actuality, the relative positions of the two domains are not the same in different crystals, because they are movable. This is shown in FIG. 4. [0046]
  • In FIG. 4, the relative movement between the two domains of the RRF in the three-dimensional structure of RRF are analyzed using the RRF crystal according to the present invention. When superimposing the lower domains in the structures of RRFs obtained from the three different crystals of RRFs, the movement of upper domains is observed. The three different crystals of RRFs are named PT1 (left side), Native1 (center) and Native2 (right side). The detergent of Native1 is depicted in FIG. 4. It is believed that the movement would be more pronounced without the detergent. The relative movement of the two domains is a troublesome factor in crystallization of RRF itself, as described above. [0047]
  • It is predicted that RRF is likely to bind to other molecules or their parts at the hydrophobic cleft formed between the movable domains. In other words, the hydrophobic cleft may act as a binding site for other molecules or their parts. This means that it is likely that the hydrophobic cleft influences the function of RRF in protein synthesis. [0048]
  • The relative movement between the two domains of RRF and the hydrophobic cleft formed by the two domains of RRF are common phenomena discovered in molecules which bind to the A-site of the ribosome such as tRNA and EF-G. This may support the fact that the relative movement between the two domains of RRF and the hydrophobic cleft are very important in the role of RRF in protein synthesis. [0049]
  • Therefore, since RRF is an essential protein for viability of bacteria(Janosi et al., 1994), RRF is an excellent target for the development of antibacterial drugs (Kaji et al., 1998) and the hydrophobic cleft of RRF is an excellent target for this purpose. Since the material binding to the hydrophobic cleft of RRF blocks the movement between the two domains of RRF, it likely blocks the ability of RRF to function in translation. Possible inhibitors can be easily screened and designed by using the three-dimensional structure of RRF according to the present invention. Ultimately, the material can be used as an RRF inhibitor and as an antibacterial agent. [0050]
  • The RRF crystal according to the present invention is obtained from the complex of RRF with decyl-β-D-maltopyranoside. This suggests that decyl-β-D-maltopyranoside is strongly bound to the hydrophobic cleft of RRF and inhibits its movement. Therefore, since decyl-β-D-maltopyranoside can inhibit the function of RRF, it can be used as an RRF inhibitor. [0051]
  • According to the three-dimensional structure of RRF analyzed from the RRF crystal with the detergent, seven carbon atoms on the alkyl chain of the detergent were found to be bound to the hydrophobic cleft of RRF. Seven of the carbon atoms from the terminus of the alkyl group are inserted into the hydrophobic cleft of RRF and the remaining three carbon atoms are outside of the cleft. The maltose group (disaccharide of 6 carbon) does not interact with the non-hydrophobic residues in the circumference of the hydrophobic cleft. Therefore, the carbon number in the hydrophobic alkyl group of the detergent binding to the cleft of RRF is preferably about 3 to 12. When the carbon number is smaller, the hydrophobic bonding is very weak. When the carbon number is larger, the part of alkyl group not binding to the cleft of RRF may act to repulse the other part of RRF. It is preferable for the detergent to have at least one group that can participate in non-hydrophobic bonding (hydrogen bonding, dipole-dipole bonding, charge-charge bonding and so on) with non-hydrophobic residues in the circumference of or near to the hydrophobic cleft of RRF. In this way, the hydrophobic alkyl group can play a role in binding to the cleft of RRF, and the non-hydrophobic group can provide the specific bonding with the non-hydrophobic amino acid residues in the circumference of or near to the cleft of RRF. In this way, the detergent can maintain high affinity binding with RRF. [0052]
  • Therefore, preferably the structure of a detergent for inhibiting the function of RRF includes an alkyl group with 3-12 carbon atoms in a straight chain and a non-hydrophobic group. The alkyl group of the detergent used in the present invention is preferably n-alkyl, but may be substituted with C1-C3 alkyl groups on at least one carbon of the straight chain. [0053]
  • Coiled Coil Domain of RRF binding to A-site of Ribosome [0054]
  • FIG. 5 provides the three-dimensional structural surface models of RRF and other molecules binding to the A-site of the ribosome (such as tRNA and EF-G). This shows the structural similarities therebetween. In the drawing, the model on the left is RRF, the center model is tRNA, and the right model is the 3,4,and 5 domains of EF-G. The white color indicates a hydrophobic surface. The red and blue colors indicate negatively and positively charged surfaces, respectively. [0055]
  • As shown in FIG. 5, the structure of RRF is similar to the structures of tRNA and EF-G, which bind to the A-site of the ribosome. In particular, the domain corresponding to the coiled coil of RRF has high structural similarity to the anticodon stem of tRNA and the domain IV of EF-G. From these and other results from several experiments, it is believed likely that the site directly binding to the A-site of the ribosome is on the coiled coil domain of RRF. [0056]
  • Therefore, the specific site binding to the A-site of the ribosome on the coiled coil domain can be identified using the three-dimensional structure of RRF according to the present invention and a material which binds to this specific site can be identified and designed. Such molecules may prevent RRF from binding to the A-site of the ribosome and can be used as an RRF inhibitor. [0057]
  • Identifying and Designing RRF inhibitors by Using the Three-Dimensional Structure of RRF [0058]
  • 1) Identifying and Designing RRF inhibitors by Using the Hydrophobic Cleft of RRF [0059]
  • The present invention relates to a method for identifying and designing complementary molecules which bind to the hydrophobic cleft of RRF by using the three-dimensional structure, and synthesizing the molecule to use as an antibiotic. The complementary molecule can be predicted to bind to the cleft of RRF physically and structurally. The binding is not likely to be covalent, but can include hydrogen bonds, ionic bonds, van der Waals and hydrophobic interactions. The processes for identifying and designing RRF inhibitors are exemplified below. [0060]
  • a. The first step is to retrieve from a database, a molecule or its part which is predicted to bind to the hydrophobic cleft of RRF in the three-dimensional structure. Alternatively a molecule can be designed which, based on its three-dimensional structure, will bind to the cleft. The binding is not likely to be covalent, but may include hydrogen bonds, ion bonds, van der Waals and hydrophobic interactions. The compound or its part may also have a special conformation which allows it to bind the cleft. The conformation is not considered in the compound itself, but in the assumption of the compound binding with the cleft. Computer modeling can perform the selection and design of the compound or fragment. Further, prior to preparing the compound, the computer modeling technique can analyze or calculate the affinity between the compound or fragment and the cleft. If the computer modeling determines that the material or its part selected from the database or designed, does not have sufficient affinity, it is less likely to have RRF inhibitory ability and will not be used subsequently. The next processes include molecule remodeling for optimizing the affinity of the molecule to the cleft. This includes modifying the structure of the compound and exchanging substituents, preparing the remodeled compound and measuring the activity of the prepared compound. In this way, the efficiency of the compound as an RRF inhibitor may be increased, because it will exclude compounds which are not likely to have inhibitor activity. If computer modeling determines that the compound has sufficient affinity for the cleft of RRF, it will be prepared and the affinity will be measured in vitro as will its activity as an inhibitor. At this point, if the dissociation constant of the complex of the compound and RRF is 100 micromoles or less, the compound may be considered as a good inhibitor candidate. When the dissociation constant is 100 micromoles, 99% of the inhibitor exists in the complex and 1% of the remainder is dissociated from the complex. The less the dissociation constant is, the more the affinity of the compound to RRF is. In paragraph b below, the design of a stronger inhibitor based on the inhibitor candidate is described. The process for calculating the affinity of the compound for RRF by the use of a computer modeling technique is to bind the compound to the hydrophobic cleft of RRF by rotating it along several planes and to fit it to the cleft by visual inspection on the computer screen. This work may be performed with modeling programs such as Insight II, Sybyl, O, Chain and Frodo. The method for inserting the compound into the hydrophobic cleft of RRF may also be performed by docking, which may be performed using modeling programs such as Quanta, Autodock, Dock, Insight II, and Sybyl. The position of the compound in the binding site may be made to be more accurate by using energy minimization or molecular dynamics with standard molecular mechanics force fields such as CHARMM and AMBER. [0061]
  • Specialized computer programs may also perform the process of selecting the compound. These include: [0062]
  • 1. GRID (Goodford, P. J., “[0063] A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules”, J. Med. Chem., 28, pp. 849-857 (1985)). GRID is available from Oxford University, Oxford, UK.
  • 2. MCSS (Miranker, A. and M. Karplus, “[0064] Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method” Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)). MCSS is available from Molecular Simulations, Burlington, Mass.
  • 3. AUTODOCK (Goodsell, D. S. and A. J. Olsen, “[0065] Automated Docking of Substates to Proteins by Simulated Annealing”, Proteins: Structure, Function and Genetics, 8, pp. 195-202 (1990)). AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.
  • 4. DOCK (Kuntz, I. D. et al., “[0066] A Geometric Approach to Macromolecule-Ligand Interactions”, J. Mol. Biol., 161, pp. 269-288 (1982). DOCK is available from University of California, San Francisco, Calif.
  • When suitable chemical compounds or fragments are selected or designed and positioned in the binding site of the hydrophobic cleft, it may be possible and desirable to assemble them into a single compound. This may be accomplished using computer modeling programs as described above as well as the following programs: [0067]
  • 1. CAVEAT (Bartlett, P. A. et al, “[0068] CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules” In “Molecular Recognition in Chemical and Biological Problems”, Special Pub., Royal Chem. Soc., 78, pp. 182-196 (1989). CAVEAT is available from the University of California, Berkeley, Calif.
  • 2. 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.). This area is reviewed in Martin, Y. C., “3[0069] D Database Searching in Drug Design”, J. Med. Chem., 35, pp. 2145-2154 (1992)).
  • 3. HOOK (available from Molecular Simulations, Burlington, Mass.). [0070]
  • Once the material is designed or selected, the affinity of the material to RRF may be calculated. For the inhibitor to be effective, it should have a low energy difference between that energy calculated before and after binding. Thus, the most efficient inhibitor should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole. The affinity of the inhibitor may also be measured by calculating the dissociation constant of the complex of RRF and the inhibitor. The dissociation constant is preferably 100 micromoles or less. The inhibitor preferably also maintains the bonding with RRF stably after binding. In order to do this, electrostatic repulsion such as charge-charge interactions, dipole-dipole and charge-dipole interactions between the inhibitor and RRF should not occur. The sum of electrostatic interaction should be neutral or give a positive effect to the enthalpy of the bonding. Examples of programs designed for calculating such affinity are as follows: Gaussian 92, revision C [M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa. © 1992]; AMBER, version 4.0 [P. A. Kollman, University of California at San Fransisco, © 1994]; QUANTA/CHARMM [Molecular Simulations, Inc., Burlington, Mass. © 1994]; and Insight II/Discover (Biosysm Technologies Inc., San Diego, Calif., © 1994). Using the lead compound selected by the method, a stronger inhibitor can be made or designed. This process will be described below. [0071]
  • b. The compound (lead compound) selected or designed as above can be changed or modified. Atoms, substituents or a part of the structure may be altered to increase the binding affinity to RRF. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. The substituted chemical compounds may then be analyzed for fit with RRF by the same computer methods described in detail, above. [0072]
  • The present invention has identified a detergent, decyl-β-D-maltopyranoside, which was used for producing an RRF crystal by binding to the hydrophobic cleft of RRF. Therefore, decyl-β-D-maltopyranoside can be used as a lead compound for the design of RRF inhibitors by performing the same computer methods described above. If needed, an appropriate substituent may be introduced into the hydrophobic chain or alkyl chain of decyl-β-D-maltopyranoside. Alternatively, the maltose group may be changed to other appropriate groups. [0073]
  • c. After the material designed by the computer method described above is prepared and bound to RRF to produce a crystal, the three-dimensional structure of the complex may be determined at high resolution (over 0.28 nm) using X-ray crystallographic methods. The information gained about the interaction between RRF and the inhibitor obtained from this study may be used to modify the inhibitor and to increase the affinity of the inhibitor to RRF. [0074]
  • d. Once the inhibitor candidate for RRF optimized by this method has been selected or designed, the actual inhibitory activity of the candidate toward RRF may be tested in vitro and in vivo. When the inhibitor candidate is determined to have inhibitory activity toward RRF, it may then be used as an inhibitor. Alternatively, it may be used for developing a more optimal inhibitor based on its structure. For example, by improving properties such as its affinity to RRF and its solubility using the computer modeling method, the inhibitory activity may be optimized. In other words, the compound may be used as a lead compound for computer remodeling to increase the properties of RRF inhibition, solubility of the compound and so on. Before the compound is tested for inhibitory activity, the dissociation constant of the complex of the inhibitor and RRF may be measured experimentally in order to confirm the affinity calculated by the computer modeling method. [0075]
  • 2) Identifying and Designing an RRF Inhibitor Based on the Three-Dimensional Structure of the RRF Domain While Binding to the A-Site of the Ribosome [0076]
  • a. Identifying the essential amino acid residues involved in binding to the ribosome. More specifically the amino acid residues in the coiled coil domain of RRF which bind to the A-site of the ribosome [0077]
  • The locations of amino acid residues in the surface of the coiled coil domain are as follows: 1, 3, 4, 6, 7, 8, 10, 11, 12, 14, 15, 17, 18, 19, 107, 108, 109, 111, 112, 114, 115, 116, 118, 119, 120, 122, 123, 125, 126, 127, 129, 130, 132, 133, 134, 136, 137, 138, 140, 141, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 156, 158, 159, 161, 162, 164, 165, 166, 167, 169, 170, 172, 173, 174, 176, 177, 179, 180, and 181. The amino acid residues are identified in the three-dimensional structure of RRF in FIG. 1 and the coordinates of the atoms in the structure of RRF are presented in table 2. [0078]
  • These atoms are considered to be involved in binding to the ribosome. RRF can be mutated by exchanging one or more of the amino acid residues on the coiled coil domain. If an [0079] E. coli possessing the mutated RRF does not grow well, grows slowly or not at all, the mutation has very likely affected the function of RRF. Specifically, the mutation has affected the ability of RRF to bind to the ribosome due to the substitution of the amino acid residue, and thus RRF cannot function in translation. This method can be used to identify amino acid residues in the original RRF which are important in the binding of RRF to the ribosome, more specifically to the A-site of the ribosome.
  • b. Identifying and designing an RRF inhibitor using the structure of the binding site in the coiled coil domain of RRF [0080]
  • Once the amino acid residues in the coiled coil domain of RRF have been identified as involved in the binding of RRF to the A-site of the ribosome, the structure of the binding site can be identified based on the three-dimensional structure of RRF. Based on the structure of the binding site, a compound such as a peptide or other compound can be screened and designed which will fit into the three-dimensional model of the binding site. For example, If a mutant which has the following residues substituted: Met-1, Ser-3 and Asp-4 has a very low activity, then these three residues are likely to be involved in the binding of RRF to the ribosome. A compound which is complementary to the structure of the binding site of RRF but includes these three residues would prevent RRF from binding to the ribosome. The complementary compound could be identified or designed from the structure of the binding site of RRF including the three residues. [0081]
  • The computer modeling method as described above can perform the identification and design of the complementary compounds. It can also be used to remodel the compound to improve the affinity or solubility, and produce an optimized compound. [0082]
  • The optimized compound may then be prepared and the inhibitory activity for RRF can be tested in vitro and in vivo. If the test confirms that the material does indeed inhibit RRF, then the material or a derivative can be used as an antibiotic. Using the method as described above, the compound identified to have inhibitory activity may be used as a lead compound to obtain an improved inhibitor. [0083]
  • In order to confirm the affinity predicted by the computer modeling method, the dissociation constant of the complex may be experimentally measured. [0084]
  • Identifying the Structure of the Complex of Inhibitor and RRF or Inhibitor and an RRF Homolog Using the Three-Dimensional Structure of RRF [0085]
  • The three-dimensional structure of RRF or one of its domains can be used to identify other crystal forms of RRF, the crystal structures of RRF homologs (those compounds having 30% amino acid similarity or more to RRF), the crystal structures of mutants of RRF or the crystal structure of a complex of RRF with another compound. Molecular replacement can be used for this. In order to identify the structure of the proteins as described above by this method, the coordinates of [0086] E. coli RRF (table 2) should be used. This method can quickly identify the unknown crystal structure. The programs used in the method are as follows:
  • CNS (Brger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T. and Warren, G. L. (1998), Crystallography & NMR system: A New software suite for macromolecular structure determination, Acta Crystallogr., D 54, 905-921). [0087]
  • CCP4 program package (Collaborative Computational Project Number 4 (1994), The CCP4 suite: programs for protein crystallography, Acta crystallogr., [0088] D 50, 760-763); It includes molecular replacement program of Amore, Polarrfn et al.
  • EPMR (Kissinger, C. R., Gehlhaar, D. K. and Fogel, D. B. (1999), Rapid automated molecular replacement by evolutionary search, Acta Crystallogr., D 55, 484-491). [0089]
  • Development of an inhibitor which mimics the coiled coil domain of RRF [0090]
  • Since the coiled coil domain of RRF is involved in the binding with the A-site of the ribosome, a polypeptide which mimics a part or all of the coiled coil domain of RRF would also bind to the A-site of the ribosome. This polypeptide will compete with RRF for binding to the A-site—making it a competitive inhibitor. As a result, it would prevent RRF from binding to the ribosome. The preparation of a polypeptide which includes the entire coiled coil domain of RRF may be performed using general biological techniques or chemical synthesis based on the secondary structure and the three-dimensional structure of the amino acid sequence. [0091]
  • Although a peptide which includes the entire coiled coil domain of RRF can be used as an inhibitor for RRF, it is not likely to be efficient due to its large size. Therefore, it will be preferable to identify an inhibitor which is an amino acid or small polypeptide which has a structure which mimics the three-dimensional coiled coil structure of the binding site of RRF to the ribosome. As described above, the binding site can be identified by testing the viability of [0092] E. coli containing mutations in the coiled coil domain of RRF. Identification and design of the compound can be performed by computer modeling as described above. Further, for example, if a three-dimensional structure formed with two amino acids is identified as binding and a second three-dimensional structure with three-amino acids is also identified as binding near the first two-amino acid compound, then each of the peptides might be used as an inhibitor for RRF. In addition, a compound formed by appropriately linking the two peptides together might also be used as an inhibitor for RRF. The latter compound would be likely to have a higher affinity to RRF than the former peptides.
  • An alternative method for developing a compound such as an amino acid or polypeptide which has the identified structure of the binding domain is to identify the compound as above, and design or synthesize it. At this point, it would not be known if this compound will bind. The inhibitory activity of this compound against RRF will then be tested in vitro or in vivo. If the compound does have inhibitory activity against RRF, it can then be used as an inhibitor. This, then is another way of identifying the structure of the binding site in the coiled coil domain. Furthermore, when two or more compounds are identified which are located close to each other on the surface of the coiled coil domain, a new compound may be designed or synthesized by linking the two compounds. The new compound may then be tested as an inhibitor of RRF. A compound composed of two or more of these compounds will have a stronger inhibitory activity than each separate compound, because it can bind more tightly to the ribosome. [0093]
    • Example 1
  • Expression and purification of [0094] E. coli RRF
  • The [0095] E. coli RRF gene was amplified by PCR using E. coli genomic DNA as a template. The PCR product was restricted and ligated into an NdeI/XhoI-restricted pET22b vector (Novagen). E. coli cells B834(DE3) were transformed with the pET22b-RRF plasmid and grown at 310° K in LB medium containing 50 μg ml−1 ampicillin. E. coli RRF was induced with 0.5 mM IPTG for 4 h. The cells were harvested at 277 K by centrifugation (5000 g for 6 min) and resuspended in buffer A (20 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1 mM PMSF). The cells were then sonicated. After a subsequent centrifugation for 30 min at 15000×g, the supernatant was applied to a 5 ml HiTrap Q column (Pharmacia) equilibrated with buffer A. The flowthrough containing the RRF protein was dialyzed against buffer B (20 mM NaH2PO4 pH 6.0, 10 mM MgCl2) and loaded onto a 5 ml HiTrap SP column (Pharmacia) equilibrated with buffer B. The protein identified in the flowthrough was concentrated by ultrafiltration (Amicon, YM 10) and loaded onto a Superdex 200 gel-filtration column (HiLoad 16/60, Pharmacia) equilibrated with buffer A containing 150 mM NaCl. The fractions containing RRF were pooled and concentrated for crystallization. The purity of RRF was checked by SDS gel electrophoresis.
    • Example 2
  • Crystallization and Structural Analysis of [0096] E. coli RRF
  • A crystal of [0097] E. coli RRF was produced using polyethylene glycol as a precipitant in the presence of the detergent, decyl-β-D-maltopyranoside (Yun et al., 2000). The procedures were as follows: The RRF protein was prepared at a concentration of 100 mg/ml in a buffer (20 mM Tris-HCl, pH 8.0, 10 MM MgCl2, 1 mM PMSF, 150 mM NaCl). The crystal growth solution had the composition: 0.1 M MES-NaOH, pH 6.5, 10% PEG 350 MME, and 12% PEG 400. 2 μl of the prepared protein solution was aliquoted onto a cover glass to form a droplet of about 2-3 mm diameter. Then 2 μl of the crystal growth solution and 0.4 μl of 18 mM decyl-β-D-maltopyranoside in turn were added to the droplet. The three solutions were not mixed other then allowing the natural mixing by diffusion. The final droplet with the three solutions was sealed, and equilibrated at 14° C. Vacuum grease was used for the sealant. Vapor diffusion of the solutions was begun in a closed system to equilibrate the solutions with the crystal growth solution and as a result, the concentration of the solution in the droplet was increased. Crystals were clustered and grew to dimensions of 0.15×0.15×1.0 mm within a week. When X-ray diffraction data from the crystal was collected and analyzed, it was found that the crystal had a unit cell of a=b=48.06 Å and c=142.27 Å, and a space group of P3121. The content of solvent was found to be 53.4%, when one molecule was in an asymmetric unit. To interpret the structure, multiple isomorphous replacement using anomalous scattering (MIRAS) and density modification such as solvent flipping was used. The programs DENZO and SCALEPACK were used for treating the X-ray diffraction data, the program “O” was used for modeling, and the program “CNS” for optimizing the model.
    TABLE 2
    The coordinates of the three-dimensional structure of RRF
    analyzed from the RRF crystal of the present invention
    Atom type Number Average B Maximum B Average Q
    Protein main chain 740 50.506 108.570 1.000
    Protein side chain 697 54.907 111.870 1.000
    Protein all atoms 1437 52.640 111.870 1.000
    Ligand/substrate 0 0.000 0.000 0.000
    Water molecules 231 61.297 96.420 1.000
    Other entities 14 65.118 79.610 1.000
    All atoms 1682 53.933 111.870 1.000
    Atom A.A. A.A.
    Atom No. Type Type No. X Y Z OCC B
    ATOM 1 CB MET 1 0.523 6.682 82.996 1.00 100.83
    ATOM 2 CG MET 1 0.660 5.557 84.005 1.00 102.28
    ATOM 3 SD MET 1 −0.459 5.752 85.397 1.00 104.69
    ATOM 4 CE MET 1 0.064 7.342 86.019 1.00 104.89
    ATOM 5 C MET 1 −0.879 7.980 81.395 1.00 98.25
    ATOM 6 O MET 1 −1.129 7.768 80.208 1.00 98.22
    ATOM 7 N MET 1 −1.317 5.554 81.781 1.00 98.73
    ATOM 8 CA MET 1 −0.883 6.836 82.409 1.00 99.14
    ATOM 9 N ILE 2 −0.586 9.186 81.876 1.00 97.02
    ATOM 10 CA ILE 2 −0.537 10.381 81.035 1.00 95.89
    ATOM 11 CB ILE 2 −0.478 11.655 81.900 1.00 95.52
    ATOM 12 CG2 ILE 2 −0.335 12.882 81.031 1.00 95.12
    ATOM 13 CG1 ILE 2 −1.750 11.762 82.734 1.00 95.60
    ATOM 14 CD1 ILE 2 −1.825 13.021 83.549 1.00 95.83
    ATOM 15 C ILE 2 0.661 10.372 80.086 1.00 95.21
    ATOM 16 O ILE 2 0.655 11.047 79.058 1.00 95.10
    ATOM 17 N SER 3 1.689 9.609 80.442 1.00 94.28
    ATOM 18 CA SER 3 2.889 9.501 79.619 1.00 93.89
    ATOM 19 CB SER 3 3.943 8.643 80.329 1.00 94.52
    ATOM 20 OG SER 3 4.245 9.152 81.617 1.00 95.52
    ATOM 21 C SER 3 2.503 8.822 78.311 1.00 92.84
    ATOM 22 O SER 3 2.949 9.203 77.229 1.00 92.71
    ATOM 23 N ASP 4 1.658 7.808 78.439 1.00 91.48
    ATOM 24 CA ASP 4 1.182 7.025 77.310 1.00 90.84
    ATOM 25 CB ASP 4 0.514 5.751 77.837 1.00 92.24
    ATOM 26 CO ASP 4 1.366 5.036 78.880 1.00 94.26
    ATOM 27 OD1 ASP 4 0.921 3.995 79.408 1.00 96.07
    ATOM 28 OD2 ASP 4 2.482 5.516 79.184 1.00 95.50
    ATOM 29 C ASP 4 0.186 7.836 76.472 1.00 88.80
    ATOM 30 O ASP 4 0.469 8.209 75.331 1.00 89.03
    ATOM 31 N ILE 5 −0.972 8.102 77.067 1.00 85.55
    ATOM 32 CA ILE 5 −2.054 8.859 76.448 1.00 81.93
    ATOM 33 CB ILE 5 −2.902 9.566 77.535 1.00 80.24
    ATOM 34 CG2 ILE 5 −3.969 10.453 76.893 1.00 79.36
    ATOM 35 CG1 ILE 5 −3.549 8.519 78.439 1.00 77.95
    ATOM 36 CD1 ILE 5 −4.442 9.097 79.495 1.00 76.67
    ATOM 37 C ILE 5 −1.686 9.895 75.379 1.00 81.10
    ATOM 38 O ILE 5 −2.162 9.803 74.247 1.00 80.52
    ATOM 39 N ARG 6 −0.857 10.879 75.718 1.00 79.50
    ATOM 40 CA ARG 6 −0.514 11.909 74.739 1.00 79.11
    ATOM 41 CB ARG 6 0.158 13.107 75.403 1.00 80.27
    ATOM 42 CG ARG 6 1.465 12.784 76.077 1.00 83.34
    ATOM 43 CD ARG 6 2.301 14.039 76.196 1.00 86.04
    ATOM 44 NE ARG 6 2.611 14.574 74.871 1.00 87.69
    ATOM 45 CZ ARG 6 3.314 15.679 74.659 1.00 87.77
    ATOM 46 NH1 ARG 6 3.780 16.374 75.692 1.00 88.33
    ATOM 47 NH2 ARG 6 3.546 16.086 73.418 1.00 85.84
    ATOM 48 C ARG 6 0.377 11.424 73.605 1.00 77.92
    ATOM 49 O ARG 6 0.228 11.866 72.467 1.00 77.98
    ATOM 50 N LYS 7 1.317 10.538 73.913 1.00 76.33
    ATOM 51 CA LYS 7 2.209 10.015 72.886 1.00 74.77
    ATOM 52 CB LYS 7 3.382 9.262 73.525 1.00 75.87
    ATOM 53 CG LYS 7 4.265 10.122 74.433 1.00 75.59
    ATOM 54 CD LYS 7 4.940 11.252 73.667 1.00 75.59
    ATOM 55 CE LYS 7 5.786 12.101 74.604 1.00 75.85
    ATOM 56 NZ LYS 7 6.601 13.132 73.900 1.00 74.95
    ATOM 57 C LYS 7 1.373 9.078 72.029 1.00 72.71
    ATOM 58 O LYS 7 1.693 8.805 70.877 1.00 72.89
    ATOM 59 N ASP 8 0.286 8.603 72.618 1.00 70.58
    ATOM 60 CA ASP 8 −0.646 7.714 71.950 1.00 69.48
    ATOM 61 CS ASP 8 −1.552 7.075 73.005 1.00 70.59
    ATOM 62 CG ASP 8 −2.393 5.940 72.459 1.00 72.83
    ATOM 63 OD1 ASP 8 −3.070 6.132 71.424 1.00 72.86
    ATOM 64 OD2 ASP 8 −2.390 4.852 73.086 1.00 72.96
    ATOM 65 C ASP 8 −1.480 8.575 70.981 1.00 68.88
    ATOM 66 O ASP 8 −1.787 8.167 69.859 1.00 68.57
    ATOM 67 N ALA 9 −1.833 9.776 71.428 1.00 66.22
    ATOM 68 CA ALA 9 −2.633 10.689 70.631 1.00 63.64
    ATOM 69 CE ALA 9 −3.115 11.854 71.494 1.00 61.48
    ATOM 70 C ALA 9 −1.885 11.215 69.413 1.00 62.96
    ATOM 71 O ALA 9 −2.455 11.287 68.323 1.00 63.12
    ATOM 72 N GLU 10 −0.617 11.580 69.575 1.00 61.05
    ATOM 73 CA GLU 10 0.124 12.114 68.437 1.00 60.56
    ATOM 74 CB GLU 10 1.385 12.848 68.888 1.00 63.50
    ATOM 75 CC GLU 10 2.407 11.994 69.599 1.00 67.84
    ATOM 76 CD GLU 10 3.728 12.731 69.774 1.00 71.48
    ATOM 77 CE1 GLU 10 4.344 13.089 68.737 1.00 72.15
    ATOM 78 CE2 GLU 10 4.141 12.958 70.938 1.00 73.41
    ATOM 79 C GLU 10 0.487 11.041 67.422 1.00 57.27
    ATOM 80 O GLU 10 0.727 11.338 66.255 1.00 55.22
    ATOM 81 N VAL 11 0.549 9.794 67.863 1.00 55.19
    ATOM 82 CA VAL 11 0.839 8.728 66.922 1.00 53.74
    ATOM 83 CS VAL 11 1.232 7.412 67.629 1.00 54.76
    ATOM 84 CC1 VAL 11 1.279 6.261 66.616 1.00 54.05
    ATOM 85 CC2 VAL 11 2.608 7.576 68.265 1.00 54.99
    ATOM 86 C VAL 11 −0.442 8.545 66.119 1.00 50.95
    ATOM 87 O VAL 11 −0.408 8.228 64.935 1.00 51.54
    ATOM 88 N ARG 12 −1.574 8.760 66.772 1.00 47.29
    ATOM 89 CA ARG 12 −2.842 8.663 66.088 1.00 45.50
    ATOM 90 CB ARG 12 −3.978 8.633 67.112 1.00 43.97
    ATOM 91 CC ARG 12 −4.126 7.256 67.793 1.00 45.79
    ATOM 92 CD ARG 12 −5.102 7.294 68.965 1.00 44.31
    ATOM 93 NE ARG 12 −5.421 5.978 69.509 1.00 44.84
    ATOM 94 CZ ARG 12 −6.083 5.776 70.653 1.00 43.11
    ATOM 95 NH1 ARG 12 −6.492 6.794 71.379 1.00 42.34
    ATOM 96 NH2 ARG 12 −6.339 4.550 71.080 1.00 45.09
    ATOM 97 C ARG 12 −2.967 9.838 65.089 1.00 43.22
    ATOM 98 O ARG 12 −3.217 9.627 63.908 1.00 40.86
    ATOM 99 N MET 13 −2.732 11.063 65.543 1.00 43.08
    ATOM 100 CA MET 13 −2.818 12.217 64.651 1.00 44.05
    ATOM 101 CB MET 13 −2.617 13.519 65.421 1.00 43.78
    ATOM 102 CG MET 13 −3.705 13.778 66.433 1.00 41.76
    ATOM 103 SD MET 13 −3.401 15.313 67.300 1.00 47.04
    ATOM 104 CE MET 13 −4.627 15.149 68.684 1.00 41.70
    ATOM 105 C MET 13 −1.848 12.179 63.482 1.00 43.79
    ATOM 106 O MET 13 −2.173 12.658 62.402 1.00 42.68
    ATOM 107 N ASP 14 −0.662 11.619 63.700 1.00 45.44
    ATOM 108 CA ASP 14 0.337 11.507 62.641 1.00 47.26
    ATOM 109 CB ASP 14 1.665 10.990 63.209 1.00 52.47
    ATOM 110 CG ASP 14 2.658 12.101 63.429 1.00 55.85
    ATOM 111 OD1 ASP 14 3.764 11.830 63.938 1.00 60.98
    ATOM 112 OD2 ASP 14 2.331 13.254 63.075 1.00 57.16
    ATOM 113 C ASP 14 −0.151 10.586 61.540 1.00 45.15
    ATOM 114 O ASP 14 −0.008 10.867 60.358 1.00 45.21
    ATOM 115 N LYE 15 −0.733 9.474 61.945 1.00 45.09
    ATOM 116 CA LYE 15 −1.264 8.529 60.999 1.00 45.94
    ATOM 117 CE LYE 15 −1.884 7.365 61.763 1.00 45.00
    ATOM 118 CG LYE 15 −2.329 6.210 60.901 1.00 49.99
    ATOM 119 CD LYE 15 −1.157 5.411 60.295 1.00 51.63
    ATOM 120 CE LYE 15 −0.326 4.634 61.339 1.00 52.42
    ATOM 121 NZ LYE 15 0.435 5.471 62.309 1.00 52.22
    ATOM 122 C LYE 15 −2.310 9.253 60.118 1.00 46.27
    ATOM 123 O LYE 15 −2.313 9.094 58.889 1.00 48.65
    ATOM 124 N CYS 16 −3.173 10.067 60.734 1.00 44.90
    ATOM 125 CA CYS 16 −4.200 10.809 59.985 1.00 44.28
    ATOM 126 CE CYE 16 −5.057 11.683 60.916 1.00 43.61
    ATOM 127 EG CYS 16 −6.022 10.834 62.181 1.00 53.86
    ATOM 128 C CYS 16 −3.604 11.730 58.908 1.00 41.26
    ATOM 129 O CYS 16 −3.971 11.676 57.729 1.00 40.04
    ATOM 130 N VAL 17 −2.697 12.596 59.324 1.00 38.60
    ATOM 131 CA VAL 17 −2.091 13.512 58.384 1.00 38.03
    ATOM 132 CB VAL 17 −1.082 14.435 59.092 1.00 39.03
    ATOM 133 CC1 VAL 17 −0.512 15.452 58.111 1.00 38.47
    ATOM 134 CC2 VAL 17 −1.766 15.144 60.257 1.00 38.78
    ATOM 135 C VAL 17 −1.394 12.737 57.269 1.00 38.04
    ATOM 136 O VAL 17 −1.530 13.074 56.095 1.00 35.00
    ATOM 137 N GLU 18 −0.670 11.684 57.635 1.00 37.93
    ATOM 138 CA GLU 18 0.034 10.913 56.636 1.00 42.29
    ATOM 139 CE GLU 18 1.057 9.962 57.315 1.00 47.92
    ATOM 140 CC GLU 18 2.136 10.774 58.105 1.00 55.13
    ATOM 141 CD GLU 18 3.403 9.987 58.501 1.00 60.44
    ATOM 142 CE1 GLU 18 4.048 9.378 57.613 1.00 60.51
    ATOM 143 CE2 GLU 18 3.779 10.009 59.704 1.00 62.88
    ATOM 144 C GLU 18 −0.935 10.205 55.687 1.00 41.27
    ATOM 145 0 GLU 18 −0.668 10.128 54.493 1.00 43.84
    ATOM 146 N ALA 19 −2.072 9.726 56.187 1.00 38.68
    ATOM 147 CA ALA 19 −3.060 9.110 55.300 1.00 37.55
    ATOM 148 CB ALA 19 −4.241 8.521 56.116 1.00 35.72
    ATOM 149 C ALA 19 −3.580 10.195 54.327 1.00 36.85
    ATOM 150 O ALA 19 −3.802 9.937 53.133 1.00 39.34
    ATOM 151 N PHE 20 −3.773 11.404 54.838 1.00 32.59
    ATOM 152 CA PEE 20 −4.234 12.498 54.010 1.00 33.05
    ATOM 153 CB PEE 20 −4.528 13.725 54.888 1.00 33.08
    ATOM 154 CC PHE 20 −4.785 14.995 54.111 1.00 32.56
    ATOM 155 CD1 PHE 20 −5.943 15.148 53.355 1.00 32.24
    ATOM 156 CD2 PHE 20 −3.868 16.037 54.148 1.00 30.14
    ATOM 157 CE1 PHE 20 −6.183 16.340 52.640 1.00 34.71
    ATOM 158 CE2 PHE 20 −4.094 17.219 53.447 1.00 34.68
    ATOM 159 CZ PHE 20 −5.257 17.375 52.689 1.00 32.40
    ATOM 160 C PHE 20 −3.162 12.830 52.965 1.00 35.46
    ATOM 161 O PHE 20 −3.460 13.017 51.772 1.00 36.29
    ATOM 162 N LYS 21 −1.911 12.916 53.405 1.00 36.65
    ATOM 163 CA LYS 21 −0.839 13.204 52.469 1.00 39.85
    ATOM 164 CB LYS 21 0.494 13.397 53.205 1.00 40.00
    ATOM 165 CC LYS 21 0.540 14.700 53.984 1.00 42.94
    ATOM 166 CD LYS 21 1.873 14.968 54.666 1.00 46.52
    ATOM 167 CE LYS 21 2.078 14.136 55.922 1.00 51.70
    ATOM 168 NZ LYS 21 2.091 12.659 55.699 1.00 56.00
    ATOM 169 C LYS 21 −0.733 12.133 51.372 1.00 42.04
    ATOM 170 O LYS 21 −0.480 12.477 50.221 1.00 42.08
    ATOM 171 N THR 22 −0.966 10.855 51.686 1.00 44.06
    ATOM 172 CA THR 22 −0.873 9.854 50.620 1.00 47.37
    ATOM 173 CB THR 22 −0.619 8.362 51.126 1.00 46.50
    ATOM 174 OG1 THR 22 −1.792 7.832 51.754 1.00 48.78
    ATOM 175 OG2 THR 22 0.524 8.314 52.105 1.00 45.25
    ATOM 176 C THR 22 −2.141 9.863 49.787 1.00 47.95
    ATOM 177 O THR 22 −2.091 9.686 48.569 1.00 50.97
    ATOM 178 N GLN 23 −3.281 10.086 50.423 1.00 50.33
    ATOM 179 CA GLN 23 −4.540 10.085 49.683 1.00 52.74
    ATOM 180 CB GLN 23 −5.705 10.316 50.637 1.00 55.39
    ATOM 181 CC GLN 23 −7.076 9.998 50.056 1.00 59.39
    ATOM 182 CD GLN 23 −8.213 10.215 51.056 1.00 61.59
    ATOM 183 OE1 GLN 23 −9.377 9.920 50.761 1.00 63.37
    ATOM 184 NE2 GLN 23 −7.881 10.734 52.239 1.00 60.88
    ATOM 185 C GLN 23 −4.549 11.159 48.598 1.00 55.03
    ATOM 186 O GLN 23 −5.064 10.948 47.487 1.00 56.03
    ATOM 187 N ILE 24 −3.946 12.299 48.923 1.00 54.26
    ATOM 188 CA ILE 24 −3.891 13.443 48.032 1.00 54.96
    ATOM 189 CB ILE 24 −3.712 14.733 48.888 1.00 56.27
    ATOM 190 CG2 ILE 24 −2.378 14.711 49.601 1.00 57.16
    ATOM 191 CG1 ILE 24 −3.785 15.976 48.019 1.00 56.46
    ATOM 192 CD1 ILE 24 −3.556 17.244 48.803 1.00 57.81
    ATOM 193 C ILE 24 −2.821 13.376 46.917 1.00 55.89
    ATOM 194 O ILE 24 −3.043 13.870 45.808 1.00 53.59
    ATOM 195 N SER 25 −1.672 12.759 47.188 1.00 56.28
    ATOM 196 CA SER 25 −0.621 12.682 46.168 1.00 57.70
    ATOM 197 CE SER 25 0.647 12.031 46.734 1.00 56.60
    ATOM 198 OG SER 25 0.421 10.671 47.061 1.00 58.84
    ATOM 199 C SER 25 −1.088 11.911 44.934 1.00 57.90
    ATOM 200 O SER 25 −0.480 12.007 43.868 1.00 59.60
    ATOM 201 N LYS 26 −2.167 11.147 45.081 1.00 57.63
    ATOM 202 CA LYS 26 −2.723 10.384 43.975 1.00 57.26
    ATOM 203 CB LYS 26 −3.825 9.454 44.474 1.00 58.98
    ATOM 204 CC LYS 26 −3.353 8.353 45.412 1.00 62.43
    ATOM 205 CD LYS 26 −4.504 7.416 45.750 1.00 64.95
    ATOM 206 CE LYS 26 −4.044 6.265 46.633 1.00 67.37
    ATOM 207 NZ LYS 26 −2.998 5.431 45.960 1.00 68.34
    ATOM 208 C LYS 26 −3.303 11.297 42.899 1.00 57.18
    ATOM 209 O LYS 26 −3.256 10.982 41.709 1.00 58.16
    ATOM 210 N ILE 27 −3.845 12.431 43.330 1.00 55.61
    ATOM 211 CA ILE 27 −4.457 13.411 42.436 1.00 54.60
    ATOM 212 CB ILE 27 −5.180 14.523 43.250 1.00 54.29
    ATOM 213 CG2 ILE 27 −5.451 15.72 642.360 1.00 54.06
    ATOM 214 CG1 ILE 27 −6.456 13.96 743.878 1.00 51.10
    ATOM 215 OD1 ILE 27 −6.215 12.81 844.784 1.00 49.52
    ATOM 216 C ILE 27 −3.515 14.096 41.444 1.00 54.74
    ATOM 217 O ILE 27 −2.557 14.776 41.825 1.00 52.96
    ATOM 218 N ARG 28 −3.810 13.925 40.163 1.00 54.44
    ATOM 219 CA ARG 28 −3.011 14.551 39.133 1.00 54.79
    ATOM 220 CD ARG 28 −3.040 13.701 37.856 1.00 57.53
    ATOM 221 CC ARG 28 −2.381 12.330 38.025 1.00 58.98
    ATOM 222 CO ARG 28 −2.325 11.578 36.707 1.00 63.90
    ATOM 223 NE ARG 28 −1.576 10.328 36.823 1.00 68.44
    ATOM 224 CZ ARG 28 −1.298 9.509 35.808 1.00 69.24
    ATOM 225 NH1 ARG 28 −1.709 9.801 34.575 1.00 69.86
    ATOM 226 NH2 ARG 28 −0.608 8.394 36.027 1.00 67.68
    ATOM 227 C ARG 28 −3.578 15.949 38.893 1.00 54.14
    ATOM 228 O ARG 28 −4.795 16.154 38.889 1.00 55.27
    ATOM 229 N THR 29 −2.698 16.915 38.705 1.00 51.89
    ATOM 230 CA THR 29 −3.134 18.285 38.506 1.00 51.42
    ATOM 231 CD THR 29 −2.414 19.187 39.502 1.00 51.85
    ATOM 232 OG1 THR 29 −1.002 19.027 39.344 1.00 51.16
    ATOM 233 CG2 THR 29 −2.786 18.798 40.924 1.00 51.25
    ATOM 234 C THR 29 −2.918 18.810 37.082 1.00 50.96
    ATOM 235 O THR 29 −3.337 19.926 36.752 1.00 50.77
    ATOM 236 N GLY 30 −2.269 17.997 36.247 1.00 49.33
    ATOM 237 CA GLY 30 −2.007 18.372 34.863 1.00 46.49
    ATOM 238 C GLY 30 −2.275 17.181 33.963 1.00 44.01
    ATOM 239 O GLY 30 −2.493 16.082 34.461 1.00 43.52
    ATOM 240 N ARG 31 −2.256 17.386 32.649 1.00 42.12
    ATOM 241 CA ARG 31 −2.520 16.301 31.703 1.00 40.50
    ATOM 242 CE ARG 31 −2.980 16.873 30.361 1.00 41.80
    ATOM 243 CG ARG 31 −4.281 17.585 30.412 1.00 41.56
    ATOM 244 CD ARG 31 −4.707 18.090 29.040 1.00 42.11
    ATOM 245 NE ARG 31 −6.018 18.713 29.146 1.00 42.16
    ATOM 246 CZ ARG 31 −7.148 18.040 29.339 1.00 42.39
    ATOM 247 NH1 ARG 31 −7.144 16.718 29.439 1.00 40.83
    ATOM 248 NH2 ARG 31 −8.279 18.699 29.490 1.00 43.78
    ATOM 249 C ARG 31 −1.350 15.347 31.440 1.00 38.97
    ATOM 250 O ARG 31 −1.540 14.209 31.015 1.00 41.05
    ATOM 251 N ALA 32 −0.143 15.812 31.698 1.00 37.59
    ATOM 252 CA ALA 32 1.059 15.033 31.454 1.00 36.56
    ATOM 253 CB ALA 32 2.278 15.811 31.987 1.00 36.73
    ATOM 254 C ALA 32 1.136 13.585 31.934 1.00 34.78
    ATOM 255 O ALA 32 0.995 13.299 33.114 1.00 37.37
    ATOM 256 N SER 33 1.397 12.678 31.001 1.00 34.58
    ATOM 257 CA SER 33 1.589 11.245 31.291 1.00 34.06
    ATOM 258 CB SER 33 0.253 10.507 31.463 1.00 36.06
    ATOM 259 OC SER 33 0.408 9.125 31.146 1.00 37.00
    ATOM 260 C SER 33 2.336 10.622 30.122 1.00 32.37
    ATOM 261 O SER 33 2.196 11.073 29.008 1.00 31.06
    ATOM 262 N PRO 34 3.192 9.621 30.377 1.00 32.68
    ATOM 263 CO PRO 34 3.664 9.098 31.670 1.00 33.36
    ATOM 264 CA PRO 34 3.924 8.970 29.285 1.00 34.44
    ATOM 265 CB PRO 34 4.871 8.021 30.029 1.00 34.92
    ATOM 266 CC PRO 34 4.085 7.690 31.284 1.00 34.26
    ATOM 267 C PRO 34 3.043 8.246 28.242 1.00 35.65
    ATOM 268 O PRO 34 3.463 8.094 27.097 1.00 36.81
    ATOM 269 N SER 35 1.829 7.824 28.613 1.00 34.52
    ATOM 270 CA SER 35 0.955 7.135 27.657 1.00 34.26
    ATOM 271 CR SER 35 0.267 5.925 28.308 1.00 33.31
    ATOM 272 OG SER 35 −0.716 6.322 29.254 1.00 36.98
    ATOM 273 C SER 35 −0.137 8.015 27.045 1.00 34.89
    ATOM 274 O SER 35 −0.975 7.514 26.308 1.00 35.76
    ATOM 275 N LEU 36 −0.120 9.313 27.334 1.00 34.17
    ATOM 276 CA LEU 36 −1.148 10.248 26.835 1.00 34.56
    ATOM 277 CS LEU 36 −0.771 11.700 27.195 1.00 35.25
    ATOM 278 CC LEU 36 −1.881 12.732 26.941 1.00 33.58
    ATOM 279 CD1 LEU 36 −3.008 12.435 27.923 1.00 35.46
    ATOM 280 CD2 LEU 36 −1.383 14.168 27.139 1.00 33.44
    ATOM 281 C LEU 36 −1.426 10.201 25.332 1.00 32.30
    ATOM 282 O LEU 36 −2.562 10.295 24.891 1.00 29.65
    ATOM 283 N LEC 37 −0.369 10.063 24.548 1.00 32.28
    ATOM 284 CA LER 37 −0.500 10.055 23.103 1.00 30.09
    ATOM 285 CR LEU 37 0.567 10.983 22.509 1.00 34.71
    ATOM 286 CG LEU 37 0.618 12.421 23.042 1.00 36.56
    ATOM 287 CD1 LEU 37 1.836 13.144 22.442 1.00 41.17
    ATOM 288 CD2 LEU 37 −0.691 13.157 22.714 1.00 35.03
    ATOM 289 C LEU 37 −0.374 8.682 22.475 1.00 27.21
    ATOM 290 O LEU 37 −0.263 8.578 21.263 1.00 26.50
    ATOM 291 N ASP 38 −0.392 7.632 23.287 1.00 28.84
    ATOM 292 CA ASP 38 −0.250 6.280 22.763 1.00 33.05
    ATOM 293 CR ASP 38 −0.363 5.233 23.889 1.00 34.31
    ATOM 294 CC ASP 38 0.851 5.229 24.810 1.00 36.92
    ATOM 295 OD1 ASP 38 0.986 4.326 25.669 1.00 39.75
    ATOM 296 OD2 ASP 38 1.681 6.140 24.679 1.00 40.38
    ATOM 297 C ASP 38 −1.228 5.942 21.630 1.00 34.44
    ATOM 298 O ASP 38 −0.852 5.245 20.694 1.00 34.98
    ATOM 299 N GLY 39 −2.452 6.465 21.713 1.00 34.31
    ATOM 300 CA GLY 39 −3.478 6.194 20.719 1.00 34.90
    ATOM 301 CO GLY 39 −3.412 6.898 19.370 1.00 35.63
    ATOM 302 O GLY 39 −4.078 6.470 18.430 1.00 36.62
    ATOM 303 N ILE 40 −2.634 7.967 19.253 1.00 35.51
    ATOM 304 CA ILE 40 −2.510 8.667 17.972 1.00 35.87
    ATOM 305 CS ILE 40 −1.503 9.862 18.082 1.00 36.81
    ATOM 306 CG2 ILE 40 −1.254 10.491 16.712 1.00 33.51
    ATOM 307 CG1 ILE 40 −2.061 10.913 19.047 1.00 37.34
    ATOM 308 CD1 ILE 40 −1.192 12.161 19.151 1.00 36.05
    ATOM 309 C ILE 40 −2.049 7.721 16.851 1.00 34.49
    ATOM 310 O ILE 40 −1.033 7.048 16.979 1.00 32.65
    ATOM 311 N VAL 41 −2.807 7.668 15.756 1.00 37.61
    ATOM 312 CA VAL 41 −2.460 6.800 14.620 1.00 37.11
    ATOM 313 CR VAL 41 −3.707 6.340 13.802 1.00 40.13
    ATOM 314 CR1 VAL 41 −4.697 5.617 14.702 1.00 41.21
    ATOM 315 CR2 VAL 41 −4.376 7.524 13.129 1.00 42.84
    ATOM 316 C VAL 41 −1.522 7.521 13.669 1.00 35.61
    ATOM 317 O VAL 41 −1.773 8.640 13.247 1.00 34.78
    ATOM 318 N VAL 42 −0.430 6.867 13.330 1.00 37.16
    ATOM 319 CA VAL 42 0.546 7.462 12.444 1.00 39.92
    ATOM 320 CB VAL 42 1.926 7.532 13.136 1.00 37.79
    ATOM 321 CG1 VAL 42 2.927 8.217 12.232 1.00 37.61
    ATOM 322 CG2 VAL 42 1.809 8.259 14.460 1.00 37.43
    ATOM 323 C VAL 42 0.689 6.621 11.182 1.00 42.15
    ATOM 324 O VAL 42 0.644 5.395 11.245 1.00 40.81
    ATOM 325 N GLU 43 0.829 7.286 10.040 1.00 44.26
    ATOM 326 CA GLU 43 1.050 6.589 8.780 1.00 47.41
    ATOM 327 CS GLU 43 0.837 7.522 7.592 1.00 50.11
    ATOM 328 CG GLU 43 −0.619 7.764 7.211 1.00 55.36
    ATOM 329 CD GLU 43 −1.297 6.497 6.686 1.00 60.26
    ATOM 330 OE1 GLU 43 −1.414 5.508 7.454 1.00 59.78
    ATOM 331 OE2 GLU 43 −1.702 6.482 5.497 1.00 63.46
    ATOM 332 C GLU 43 2.496 6.121 8.800 1.00 48.82
    ATOM 333 O GLU 43 3.420 6.928 8.699 1.00 48.94
    ATOM 334 N TYR 44 2.691 4.817 8.943 1.00 50.69
    ATOM 335 CA TYR 44 4.032 4.237 8.999 1.00 52.56
    ATOM 336 CB TYR 44 4.193 3.521 10.337 1.00 51.42
    ATOM 337 CC TYR 44 5.560 2.957 10.626 1.00 51.48
    ATOM 338 CD1 TYR 44 6.670 3.791 10.773 1.00 49.46
    ATOM 339 CE1 TYR 44 7.919 3.268 11.107 1.00 49.00
    ATOM 340 CD2 TYR 44 5.739 1.581 10.811 1.00 50.45
    ATOM 341 CR2 TYR 44 6.982 1.055 11.143 1.00 49.22
    ATOM 342 CZ TYR 44 8.064 1.898 11.295 1.00 48.06
    ATOM 343 OH TYR 44 9.268 1.366 11.682 1.00 47.29
    ATOM 344 C TYR 44 4.165 3.263 7.832 1.00 54.71
    ATOM 345 O TYR 44 3.643 2.147 7.873 1.00 55.85
    ATOM 346 N TYR 45 4.864 3.708 6.790 1.00 58.86
    ATOM 347 CA TYR 45 5.067 2.941 5.552 1.00 60.22
    ATOM 348 CB TYR 45 6.152 1.879 5.721 1.00 60.58
    ATOM 349 CC TYR 45 7.497 2.434 6.123 1.00 60.52
    ATOM 350 CD1 TYR 45 7.701 2.944 7.400 1.00 60.80
    ATOM 351 CE1 TYR 45 8.937 3.464 7.780 1.00 61.27
    ATOM 352 CD2 TYR 45 8.564 2.458 5.226 1.00 59.56
    ATOM 353 CE2 TYR 45 9.801 2.977 5.596 1.00 61.01
    ATOM 354 CZ TYR 45 9.979 3.482 6.881 1.00 60.45
    ATOM 355 OH TYR 45 11.185 4.030 7.276 1.00 62.38
    ATOM 356 C TYR 45 3.782 2.280 5.076 1.00 61.45
    ATOM 357 O TYR 45 3.724 1.064 4.912 1.00 61.25
    ATOM 358 N GLY 46 2.751 3.100 4.873 1.00 64.05
    ATOM 359 CA GLY 46 1.461 2.608 4.407 1.00 65.41
    ATOM 360 C GLY 46 0.458 2.193 5.473 1.00 66.35
    ATOM 361 O GLY 46 −0.737 2.509 5.368 1.00 66.56
    ATOM 362 N THR 47 0.952 1.501 6.501 1.00 66.38
    ATOM 363 CA THR 47 0.126 0.982 7.594 1.00 65.82
    ATOM 364 CB THR 47 0.711 −0.347 8.135 1.00 67.46
    ATOM 365 OG1 THR 47 2.011 −0.101 8.693 1.00 69.00
    ATOM 366 OG2 THR 47 0.842 −1.375 7.012 1.00 67.57
    ATOM 367 C THR 47 −0.058 1.920 8.789 1.00 63.78
    ATOM 368 O THR 47 0.912 2.391 9.393 1.00 63.80
    ATOM 369 N PRO 48 −1.317 2.208 9.145 1.00 61.42
    ATOM 370 CD PRO 48 −2.587 1.799 8.522 1.00 59.86
    ATOM 371 CA PRO 48 −1.577 3.092 10.286 1.00 58.41
    ATOM 372 CE PRO 48 −3.097 3.296 10.217 1.00 58.67
    ATOM 373 CC PRO 48 −3.403 3.068 8.701 1.00 59.94
    ATOM 374 C PRO 48 −1.137 2.360 11.550 1.00 54.30
    ATOM 375 O PRO 48 −1.545 1.228 11.790 1.00 53.93
    ATOM 376 N THR 49 −0.287 2.994 12.344 1.00 49.82
    ATOM 377 CA THR 49 0.191 2.363 13.560 1.00 45.54
    ATOM 378 CE THR 49 1.626 1.811 13.384 1.00 45.94
    ATOM 379 OG1 THR 49 2.548 2.895 13.261 1.00 51.01
    ATOM 380 OG2 THR 49 1.724 0.975 12.129 1.00 44.44
    ATOM 381 C THR 49 0.167 3.344 14.723 1.00 41.67
    ATOM 382 O THR 49 0.462 4.526 14.563 1.00 39.81
    ATOM 383 N PRO 50 −0.207 2.861 15.915 1.00 38.96
    ATOM 384 CD PRO 50 −0.608 1.492 16.275 1.00 36.47
    ATOM 385 CA PRO 50 −0.258 3.731 17.087 1.00 35.39
    ATOM 386 CB PRO 50 −0.768 2.787 18.189 1.00 35.03
    ATOM 387 CG PRO 50 −1.589 1.770 17.391 1.00 37.74
    ATOM 388 C PRO 50 1.134 4.282 17.372 1.00 34.29
    ATOM 389 O PRO 50 2.132 3.566 17.222 1.00 32.88
    ATOM 390 N LEO 51 1.177 5.553 17.777 1.00 32.65
    ATOM 391 CA LEU 51 2.408 6.261 18.106 1.00 32.71
    ATOM 392 CB LEU 51 2.057 7.654 18.627 1.00 32.91
    ATOM 393 CG LEU 51 3.183 8.543 19.140 1.00 32.51
    ATOM 394 CD1 LEU 51 4.317 8.566 18.117 1.00 35.14
    ATOM 395 CD2 LEU 51 2.644 9.950 19.354 1.00 34.75
    ATOM 396 C LEU 51 3.259 5.527 19.146 1.00 35.19
    ATOM 397 O LEU 51 4.500 5.620 19.153 1.00 32.93
    ATOM 398 N ARG 52 2.573 4.817 20.036 1.00 35.13
    ATOM 399 CA ARG 52 3.205 4.055 21.102 1.00 35.65
    ATOM 400 CB ARG 52 2.122 3.263 21.853 1.00 35.45
    ATOM 401 CG ARG 52 2.629 2.303 22.934 1.00 37.41
    ATOM 402 CO ARG 52 1.456 1.582 23.582 1.00 39.06
    ATOM 403 NE ARG 52 0.576 1.021 22.547 1.00 44.41
    ATOM 404 CZ ARG 52 0.722 −0.168 21.949 1.00 44.42
    ATOM 405 NE1 ARG 52 1.726 −0.983 22.266 1.00 41.83
    ATOM 406 NH2 ARG 52 −0.138 −0.526 21.005 1.00 42.08
    ATOM 407 C ARG 52 4.299 3.103 20.586 1.00 34.33
    ATOM 408 O ARG 52 5.304 2.914 21.241 1.00 35.09
    ATOM 409 N GLN 53 4.092 2.519 19.417 1.00 34.91
    ATOM 410 CA GLN 53 5.049 1.572 18.837 1.00 38.97
    ATOM 411 CE GLN 53 4.322 0.539 17.959 1.00 40.17
    ATOM 412 CC GLN 53 3.235 −0.225 18.663 1.00 46.58
    ATOM 413 CD GLN 53 2.466 −1.151 17.736 1.00 50.36
    ATOM 414 OE1 GLN 53 1.897 −0.727 16.727 1.00 50.89
    ATOM 415 NE2 GLN 53 2.429 −2.431 18.090 1.00 55.52
    ATOM 416 C GLN 53 6.111 2.244 17.966 1.00 39.42
    ATOM 417 O GLN 53 6.975 1.577 17.447 1.00 36.91
    ATOM 418 N LEU 54 6.033 3.560 17.808 1.00 40.51
    ATOM 419 CA LEU 54 6.967 4.272 16.963 1.00 40.80
    ATOM 420 CB LEU 54 6.179 4.999 15.868 1.00 39.66
    ATOM 421 CC LEU 54 5.261 4.086 15.042 1.00 44.30
    ATOM 422 CD1 LEU 54 4.362 4.929 14.106 1.00 42.27
    ATOM 423 CD2 LEU 54 6.102 3.085 14.243 1.00 40.97
    ATOM 424 C LEU 54 7.823 5.269 17.739 1.00 40.78
    ATOM 425 O LEU 54 8.772 5.817 17.214 1.00 41.97
    ATOM 426 N ALA 55 7.506 5.510 18.998 1.00 40.71
    ATOM 427 CA ALA 55 8.285 6.485 19.734 1.00 40.64
    ATOM 428 CB ALA 55 7.855 7.889 19.332 1.00 38.83
    ATOM 429 C ALA 55 8.180 6.352 21.232 1.00 40.46
    ATOM 430 O ALA 55 7.256 5.747 21.759 1.00 40.59
    ATOM 431 N SER 56 9.166 6.927 21.898 1.00 41.32
    ATOM 432 CA SER 56 9.243 6.982 23.346 1.00 41.03
    ATOM 433 CE SER 56 10.715 6.932 23.771 1.00 44.77
    ATOM 434 CC SER 56 10.860 7.109 25.169 1.00 49.29
    ATOM 435 C SER 56 8.635 8.339 23.718 1.00 39.72
    ATOM 436 O SER 56 9.024 9.365 23.151 1.00 37.62
    ATOM 437 N VAL 57 7.667 8.350 24.634 1.00 40.18
    ATOM 438 CA VAL 57 7.041 9.605 25.059 1.00 38.81
    ATOM 439 CB VAL 57 5.503 9.601 24.817 1.00 38.08
    ATOM 440 CG1 VAL 57 4.885 10.935 25.240 1.00 35.66
    ATOM 441 CG2 VAL 57 5.218 9.368 23.360 1.00 34.50
    ATOM 442 C VAL 57 7.334 9.821 26.530 1.00 41.07
    ATOM 443 O VAL 57 6.827 9.096 27.386 1.00 43.17
    ATOM 444 N THR 58 8.159 10.828 26.814 1.00 42.76
    ATOM 445 CA THR 58 8.577 11.160 28.173 1.00 44.28
    ATOM 446 CB THR 58 10.109 11.316 28.251 1.00 44.97
    ATOM 447 OG1 THR 58 10.730 10.029 28.151 1.00 50.84
    ATOM 448 OG2 THR 58 10.522 11.948 29.563 1.00 50.51
    ATOM 449 C THR 58 7.953 12.444 28.670 1.00 46.01
    ATOM 450 O THR 58 7.716 13.373 27.888 1.00 47.72
    ATOM 451 N VAL 59 7.689 12.516 29.971 1.00 45.39
    ATOM 452 CA VAL 59 7.107 13.736 30.522 1.00 45.59
    ATOM 453 CB VAL 59 6.336 13.468 31.829 1.00 42.70
    ATOM 454 CG1 VAL 59 5.726 14.768 32.327 1.00 42.59
    ATOM 455 CG2 VAL 59 5.272 12.408 31.613 1.00 41.35
    ATOM 456 C VAL 59 8.218 14.753 30.809 1.00 48.24
    ATOM 457 O VAL 59 8.936 14.645 31.801 1.00 44.55
    ATOM 458 N GLU 60 8.369 15.736 29.933 1.00 48.24
    ATOM 459 CA GLU 60 9.400 16.740 30.136 1.00 50.27
    ATOM 460 CE GLU 60 9.559 17.588 28.868 1.00 50.29
    ATOM 461 CC GLU 60 10.513 18.765 28.996 1.00 53.37
    ATOM 462 CD GLU 60 10.795 19.442 27.659 1.00 53.53
    ATOM 463 OE1 GLU 60 11.657 18.958 26.912 1.00 56.23
    ATOM 464 OE2 GLU 60 10.145 20.453 27.336 1.00 55.58
    ATOM 465 C GLU 60 9.004 17.575 31.357 1.00 51.63
    ATOM 466 O GLU 60 9.825 17.863 32.216 1.00 51.46
    ATOM 467 N ASP 61 7.730 17.927 31.445 1.00 53.42
    ATOM 468 CA ASP 61 7.225 18.694 32.579 1.00 56.91
    ATOM 469 OB ASP 61 7.567 20.176 32.418 1.00 57.87
    ATOM 470 CG ASP 61 7.065 20.736 31.115 1.00 59.93
    ATOM 471 OD1 ASP 61 7.636 20.363 30.059 1.00 60.62
    ATOM 472 OD2 ASP 61 6.085 21.520 31.144 1.00 61.00
    ATOM 473 C ASP 61 5.701 18.531 32.694 1.00 57.24
    ATOM 474 O ASP 61 5.055 18.036 31.775 1.00 59.19
    ATOM 475 N SER 62 5.146 18.967 33.821 1.00 57.11
    ATOM 476 CA SER 62 3.714 18.893 34.120 1.00 56.21
    ATOM 477 CB SER 62 3.404 19.839 35.267 1.00 58.84
    ATOM 478 OG SER 62 3.730 21.172 34.891 1.00 61.16
    ATOM 479 C SER 62 2.727 19.186 32.980 1.00 54.83
    ATOM 480 O SER 62 1.606 18.675 32.988 1.00 54.24
    ATOM 481 N ARG 63 3.115 20.019 32.022 1.00 52.32
    ATOM 482 CA ARG 63 2.214 20.326 30.920 1.00 51.50
    ATOM 483 CE ARG 63 1.575 21.711 31.112 1.00 53.61
    ATOM 484 CG ARG 63 2.516 22.912 31.044 1.00 57.29
    ATOM 485 CD ARG 63 1.906 24.083 31.836 1.00 60.21
    ATOM 486 NE ARG 63 2.647 25.338 31.717 1.00 62.28
    ATOM 487 CZ ARG 63 2.565 26.160 30.675 1.00 65.53
    ATOM 488 NE1 ARG 63 1.767 25.860 29.651 1.00 66.70
    ATOM 489 NH2 ARG 63 3.272 27.289 30.657 1.00 65.73
    ATOM 490 C ARG 63 2.893 20.241 29.562 1.00 48.65
    ATOM 491 O ARG 63 2.458 20.872 28.606 1.00 49.55
    ATOM 492 N THR 64 3.946 19.438 29.473 1.00 44.74
    ATOM 493 CA THR 64 4.659 19.298 28.215 1.00 43.06
    ATOM 494 CB THR 64 5.827 20.305 28.134 1.00 42.31
    ATOM 495 OG1 THR 64 5.337 21.616 28.443 1.00 46.00
    ATOM 496 OG2 THR 64 6.440 20.321 26.726 1.00 41.82
    ATOM 497 C THR 64 5.215 17.884 27.983 1.00 41.37
    ATOM 498 O THR 64 5.806 17.270 28.888 1.00 38.27
    ATOM 499 N LED 65 5.010 17.363 26.777 1.00 37.51
    ATOM 500 CA LED 65 5.540 16.050 26.464 1.00 38.11
    ATOM 501 CB LED 65 4.477 15.102 25.875 1.00 34.91
    ATOM 502 CC LED 65 3.255 14.797 26.745 1.00 35.31
    ATOM 503 CD1 LEU 65 2.412 13.721 26.098 1.00 33.78
    ATOM 504 CD2 LEU 65 3.712 14.367 28.108 1.00 34.37
    ATOM 505 C LEU 65 6.669 16.170 25.473 1.00 38.28
    ATOM 506 O LEU 65 6.685 17.051 24.614 1.00 38.59
    ATOM 507 N LYS 66 7.615 15.254 25.613 1.00 40.15
    ATOM 508 CA LYS 66 8.774 15.161 24.743 1.00 40.02
    ATOM 509 CE LYS 66 10.043 15.189 25.588 1.00 45.80
    ATOM 510 CO LYS 66 11.202 14.454 24.977 1.00 51.87
    ATOM 511 CD LYS 66 12.272 14.216 26.026 1.00 56.23
    ATOM 512 CE LYS 66 13.346 13.286 25.476 1.00 59.78
    ATOM 513 NZ LYS 66 14.455 13.197 26.441 1.00 60.71
    ATOM 514 C LYS 66 8.609 13.823 24.031 1.00 37.57
    ATOM 515 O LYS 66 8.606 12.770 24.668 1.00 33.81
    ATOM 516 N ILE 67 8.474 13.871 22.715 1.00 36.18
    ATOM 517 CA ILE 67 8.313 12.666 21.923 1.00 37.12
    ATOM 518 CB ILE 67 7.241 12.841 20.853 1.00 36.65
    ATOM 519 CG2 ILE 67 7.057 11.546 20.108 1.00 39.63
    ATOM 520 CG1 ILE 67 5.911 13.234 21.473 1.00 34.46
    ATOM 521 CD1 ILE 67 4.873 13.466 20.423 1.00 35.05
    ATOM 522 C ILE 67 9.624 12.276 21.246 1.00 39.04
    ATOM 523 O ILE 67 10.195 13.035 20.466 1.00 39.66
    ATOM 524 N ASN 68 10.086 11.075 21.539 1.00 40.32
    ATOM 525 CA ASN 68 11.331 10.606 20.975 1.00 41.37
    ATOM 526 CB ASN 68 12.197 10.115 22.119 1.00 44.76
    ATOM 527 CC ASN 68 13.584 9.842 21.691 1.00 47.81
    ATOM 528 OD1 ASN 68 14.155 10.636 20.951 1.00 52.33
    ATOM 529 ND2 ASN 68 14.162 8.727 22.155 1.00 50.81
    ATOM 530 C ASN 68 11.107 9.511 19.920 1.00 40.11
    ATOM 531 O ASN 68 11.005 8.331 20.248 1.00 38.50
    ATOM 532 N VAL 69 11.031 9.922 18.657 1.00 38.66
    ATOM 533 CA VAL 69 10.794 9.008 17.539 1.00 39.78
    ATOM 534 CB VAL 69 10.383 9.806 16.277 1.00 38.68
    ATOM 535 CG1 VAL 69 9.997 8.867 15.165 1.00 39.74
    ATOM 536 CG2 VAL 69 9.221 10.733 16.600 1.00 37.97
    ATOM 537 C VAL 69 12.060 8.177 17.278 1.00 39.57
    ATOM 538 O VAL 69 13.156 8.741 17.193 1.00 40.97
    ATOM 539 N PHE 70 11.909 6.855 17.146 1.00 36.02
    ATOM 540 CA PHE 70 13.059 5.953 16.968 1.00 35.77
    ATOM 541 CB PHE 70 12.660 4.495 17.287 1.00 36.22
    ATOM 542 CG PHE 70 12.026 4.311 18.644 1.00 34.76
    ATOM 543 CD1 PHE 70 12.692 4.693 19.803 1.00 32.72
    ATOM 544 CD2 PHE 70 10.764 3.720 18.757 1.00 36.32
    ATOM 545 CE1 PHE 70 12.119 4.492 21.050 1.00 34.27
    ATOM 546 CE2 PHE 70 10.176 3.514 20.003 1.00 32.75
    ATOM 547 CZ PHE 70 10.857 3.901 21.157 1.00 34.27
    ATOM 548 C PHE 70 13.731 5.996 15.597 1.00 35.41
    ATOM 549 O PHE 70 14.947 5.811 15.474 1.00 35.74
    ATOM 550 N ASP 71 12.924 6.209 14.574 1.00 34.31
    ATOM 551 CA ASP 71 13.386 6.317 13.206 1.00 36.54
    ATOM 552 CE ASP 71 12.552 5.406 12.299 1.00 37.50
    ATOM 553 CG ASP 71 12.906 5.536 10.837 1.00 42.15
    ATOM 554 OD1 ASP 71 13.497 6.580 10.444 1.00 42.99
    ATOM 555 OD2 ASP 71 12.556 4.596 10.071 1.00 43.58
    ATOM 556 C ASP 71 13.156 7.797 12.855 1.00 36.95
    ATOM 557 O ASP 71 12.008 8.249 12.711 1.00 34.20
    ATOM 558 N ARG 72 14.261 8.530 12.743 1.00 37.72
    ATOM 559 CA ARG 72 14.260 9.957 12.453 1.00 41.91
    ATOM 560 CE ARG 72 15.714 10.444 12.340 1.00 44.20
    ATOM 561 CC ARG 72 15.89 711.931 12.101 1.00 50.61
    ATOM 562 CD ARG 72 17.379 12.304 12.148 1.00 54.03
    ATOM 563 NE ARG 72 17.935 12.179 13.495 1.00 58.08
    ATOM 564 CZ ARG 72 19.241 12.176 13.773 1.00 61.41
    ATOM 565 NH1 ARG 72 20.136 12.293 12.793 1.00 60.31
    ATOM 566 NH2 ARG 72 19.658 12.056 15.033 1.00 60.96
    ATOM 567 C ARG 72 13.447 10.317 11.205 1.00 42.06
    ATOM 568 O ARG 72 12.784 11.349 11.166 1.00 41.02
    ATOM 569 N SER 73 13.459 9.452 10.199 1.00 42.69
    ATOM 570 CA SER 73 12.700 9.719 8.991 1.00 41.82
    ATOM 571 CB SER 73 13.007 8.655 7.935 1.00 44.65
    ATOM 572 OG SER 73 12.665 7.354 8.392 1.00 47.00
    ATOM 573 C SER 73 11.190 9.779 9.233 1.00 42.72
    ATOM 574 O SER 73 10.449 10.336 8.417 1.00 42.14
    ATOM 575 N MET 74 10.730 9.199 10.338 1.00 41.95
    ATOM 576 CA MET 74 9.304 9.205 10.666 1.00 44.14
    ATOM 577 CB MET 74 8.932 7.959 11.479 1.00 44.72
    ATOM 578 CC MET 74 8.928 6.674 10.705 1.00 45.64
    ATOM 579 SD MET 74 7.787 6.778 9.304 1.00 49.27
    ATOM 580 CE MET 74 8.965 7.228 7.973 1.00 49.04
    ATOM 581 C MET 74 8.819 10.433 11.442 1.00 43.59
    ATOM 582 O MET 74 7.612 10.629 11.605 1.00 43.28
    ATOM 583 N SER 75 9.750 11.237 11.939 1.00 42.08
    ATOM 584 CA SER 75 9.381 12.406 12.715 1.00 42.19
    ATOM 585 CE SER 75 10.621 13.208 13.085 1.00 42.09
    ATOM 586 CC SER 75 10.281 14.280 13.941 1.00 46.20
    ATOM 587 C SER 75 8.376 13.301 11.991 1.00 42.07
    ATOM 588 O SER 75 7.364 13.698 12.574 1.00 40.55
    ATOM 589 N PRO 76 8.650 13.666 10.728 1.00 41.74
    ATOM 590 CD PRO 76 9.809 13.391 9.861 1.00 41.37
    ATOM 591 CA PRO 76 7.688 14.518 10.021 1.00 40.98
    ATOM 592 CB PRO 76 8.284 14.610 8.622 1.00 40.75
    ATOM 593 CC PRO 76 9.773 14.597 8.928 1.00 41.52
    ATOM 594 C PRO 76 6.283 13.904 10.027 1.00 40.31
    ATOM 595 O PRO 76 5.293 14.595 10.298 1.00 40.54
    ATOM 596 N ALA 77 6.207 12.607 9.737 1.00 38.47
    ATOM 597 CA ALA 77 4.926 11.897 9.711 1.00 39.23
    ATOM 598 CB ALA 77 5.130 10.451 9.209 1.00 39.21
    ATOM 599 C ALA 77 4.260 11.887 11.097 1.00 37.86
    ATOM 600 O ALA 77 3.042 11.977 11.204 1.00 37.27
    ATOM 601 N VAL 78 5.067 11.769 12.148 1.00 35.44
    ATOM 602 CA VAL 78 4.542 11.754 13.495 1.00 34.88
    ATOM 603 CE VAL 78 5.647 11.436 14.541 1.00 33.08
    ATOM 604 CG1 VAL 78 5.135 11.747 15.952 1.00 27.26
    ATOM 605 CG2 VAL 78 6.021 9.967 14.467 1.00 34.28
    ATOM 606 C VAL 78 3.942 13.107 13.812 1.00 36.71
    ATOM 607 O VAL 78 2.886 13.205 14.438 1.00 35.21
    ATOM 608 N GLU 79 4.641 14.153 13.388 1.00 38.95
    ATOM 609 CA GLU 79 4.193 15.507 13.628 1.00 41.23
    ATOM 610 CE GLU 79 5.248 16.517 13.183 1.00 42.75
    ATOM 611 CC GLU 79 4.774 17.950 13.309 1.00 49.69
    ATOM 612 CD GLU 79 5.928 18.925 13.461 1.00 53.09
    ATOM 613 OE1 GLU 79 6.899 18.828 12.664 1.00 56.30
    ATOM 614 OE2 GLU 79 5.858 19.784 14.370 1.00 50.54
    ATOM 615 C GLU 79 2.874 15.764 12.937 1.00 40.64
    ATOM 616 O GLU 79 1.996 16.380 13.517 1.00 43.12
    ATOM 617 N LYS 80 2.723 15.275 11.714 1.00 41.68
    ATOM 618 CA LYS 80 1.465 15.442 11.002 1.00 43.65
    ATOM 619 CE LYS 80 1.586 14.991 9.542 1.00 47.34
    ATOM 620 CC LYS 80 2.415 15.924 8.634 1.00 51.49
    ATOM 621 CD LYS 80 1.969 17.397 8.749 1.00 54.11
    ATOM 622 CE LYS 80 2.242 18.208 7.450 1.00 58.81
    ATOM 623 NZ LYS 80 3.659 18.188 6.930 1.00 59.99
    ATOM 624 C LYS 80 0.356 14.636 11.674 1.00 43.12
    ATOM 625 O LYS 80 −0.800 15.077 11.748 1.00 43.15
    ATOM 626 N ALA 81 0.708 13.451 12.158 1.00 40.38
    ATOM 627 CA ALA 81 −0.257 12.598 12.824 1.00 38.88
    ATOM 628 CE ALA 81 0.387 11.255 13.201 1.00 36.34
    ATOM 629 C ALA 81 −0.785 13.302 14.072 1.00 39.56
    ATOM 630 O ALA 81 −1.964 13.183 14.404 1.00 41.23
    ATOM 631 N ILE 82 0.074 14.045 14.766 1.00 39.16
    ATOM 632 CA ILE 82 −0.380 14.720 15.971 1.00 38.15
    ATOM 633 CE ILE 82 0.780 15.267 16.803 1.00 35.54
    ATOM 634 CG2 ILE 82 0.241 16.060 17.965 1.00 33.27
    ATOM 635 CG1 ILE 82 1.626 14.111 17.351 1.00 35.88
    ATOM 636 CD1 ILE 82 2.878 14.562 18.122 1.00 36.02
    ATOM 637 C ILE 82 −1.326 15.860 15.620 1.00 40.83
    ATOM 638 O ILE 82 −2.373 16.019 16.231 1.00 39.39
    ATOM 639 N MET 83 −0.965 16.660 14.633 1.00 42.37
    ATOM 640 CA MET 83 −1.856 17.733 14.268 1.00 44.18
    ATOM 641 CE MET 83 −1.238 18.589 13.164 1.00 47.58
    ATOM 642 CG MET 83 0.119 19.148 13.565 1.00 52.70
    ATOM 643 SD MET 83 0.871 20.375 12.470 1.00 58.98
    ATOM 644 CE MET 83 0.820 19.513 10.881 1.00 57.30
    ATOM 645 C MET 83 −3.169 17.128 13.806 1.00 43.27
    ATOM 646 O MET 83 −4.237 17.593 14.206 1.00 44.19
    ATOM 647 N ALA 84 −3.081 16.044 13.034 1.00 41.67
    ATOM 648 CA ALA 84 −4.260 15.376 12.456 1.00 40.91
    ATOM 649 CE ALA 84 −3.821 14.343 11.427 1.00 39.91
    ATOM 650 C ALA 84 −5.264 14.737 13.404 1.00 40.62
    ATOM 651 O ALA 84 −6.427 14.590 13.059 1.00 40.16
    ATOM 652 N SER 85 −4.823 14.338 14.586 1.00 40.70
    ATOM 653 CA SER 85 −5.729 13.716 15.542 1.00 39.44
    ATOM 654 CB SER 85 −4.956 13.233 16.774 1.00 37.78
    ATOM 655 OG SER 85 −4.436 14.330 17.503 1.00 36.38
    ATOM 656 C SER 85 −6.797 14.713 15.976 1.00 38.77
    ATOM 657 O SER 85 −7.799 14.333 16.543 1.00 38.69
    ATOM 658 N ASP 86 −6.550 15.993 15.724 1.00 40.30
    ATOM 659 CA ASP 86 −7.477 17.073 16.070 1.00 39.72
    ATOM 660 CB ASP 86 −8.750 16.964 15.244 1.00 40.60
    ATOM 661 CG ASP 86 −9.690 18.137 15.462 1.00 43.77
    ATOM 662 OD1 ASP 86 −9.259 19.302 15.305 1.00 45.04
    ATOM 663 OD2 ASP 86 −10.872 17.896 15.782 1.00 45.78
    ATOM 664 C ASP 86 −7.816 17.123 17.548 1.00 40.91
    ATOM 665 O ASP 86 −8.980 17.131 17.948 1.00 40.62
    ATOM 666 N LEU 87 −6.768 17.161 18.355 1.00 41.54
    ATOM 667 CA LEU 87 −6.892 17.230 19.796 1.00 44.40
    ATOM 668 CB LEU 87 −6.028 16.146 20.446 1.00 44.35
    ATOM 669 CG LEU 87 −6.395 14.711 20.057 1.00 47.40
    ATOM 670 CD1 LEU 87 −5.385 13.701 20.596 1.00 45.31
    ATOM 671 CD2 LEU 87 −7.791 14.427 20.595 1.00 46.71
    ATOM 672 C LEU 87 −6.452 18.614 20.273 1.00 46.06
    ATOM 673 O LEU 87 −6.292 18.840 21.468 1.00 46.97
    ATOM 674 N GLY 88 −6.245 19.527 19.324 1.00 48.12
    ATOM 675 CA GLY 88 −5.841 20.883 19.654 1.00 47.94
    ATOM 676 C GLY 88 −4.377 20.975 20.005 1.00 47.52
    ATOM 677 O GLY 88 −3.891 22.030 20.404 1.00 47.24
    ATOM 673 N LEU 89 −3.676 19.858 19.853 1.00 48.46
    ATOM 679 CA LEU 89 −2.252 19.788 20.159 1.00 48.30
    ATOM 680 CB LEU 89 −1.822 18.334 20.374 1.00 47.12
    ATOM 681 CC LEU 89 −2.643 17.495 21.352 1.00 48.13
    ATOM 682 CD1 LEU 89 −2.063 16.072 21.451 1.00 45.92
    ATOM 683 CD2 LEU 89 −2.650 18.170 22.701 1.00 49.76
    ATOM 684 C LEU 89 −1.400 20.404 19.043 1.00 49.93
    ATOM 685 O LEU 89 −1.463 19.976 17.877 1.00 49.90
    ATOM 686 N ASN 90 −0.611 21.410 19.423 1.00 49.86
    ATOM 687 CA ASN 90 0.292 22.120 18.523 1.00 50.18
    ATOM 688 CB ASN 90 0.305 23.625 18.852 1.00 51.76
    ATOM 689 CC ASN 90 −1.056 24.282 18.689 1.00 51.96
    ATOM 690 OD1 ASN 90 −1.630 24.289 17.604 1.00 52.89
    ATOM 691 OD2 ASN 90 −1.576 24.845 19.774 1.00 55.10
    ATOM 692 C ASN 90 1.693 21.560 18.747 1.00 49.01
    ATOM 693 O ASN 90 2.419 22.030 19.617 1.00 47.33
    ATOM 694 N PRO 91 2.080 20.534 17.981 1.00 49.04
    ATOM 695 CD PRO 91 1.310 19.836 16.941 1.00 47.70
    ATOM 696 CA PRO 91 3.405 19.921 18.109 1.00 50.17
    ATOM 697 CB PRO 91 3.313 18.750 17.140 1.00 49.46
    ATOM 698 CG PRO 91 2.407 19.315 16.064 1.00 47.73
    ATOM 699 C PRO 91 4.519 20.901 17.734 1.00 52.02
    ATOM 700 O PRO 91 4.437 21.584 16.716 1.00 49.73
    ATOM 701 N ASN 92 5.551 20.978 18.569 1.00 55.38
    ATOM 702 CA ASN 92 6.677 21.876 18.316 1.00 56.64
    ATOM 703 CB ASN 92 6.907 22.779 19.527 1.00 57.88
    ATOM 704 CG ASN 92 5.868 23.895 19.624 1.00 61.72
    ATOM 705 OD1 ASN 92 5.687 24.507 20.679 1.00 63.08
    ATOM 706 OD2 ASN 92 5.195 24.178 18.509 1.00 61.74
    ATOM 707 C ASN 92 7.901 21.053 17.999 1.00 56.71
    ATOM 708 O ASN 92 8.540 20.511 18.889 1.00 56.47
    ATOM 709 N SER 93 8.212 20.957 16.711 1.00 58.68
    ATOM 710 CA SER 93 9.340 20.163 16.241 1.00 61.01
    ATOM 711 CB SER 93 9.026 19.614 14.846 1.00 60.28
    ATOM 712 CG SER 93 10.014 18.692 14.424 1.00 60.68
    ATOM 713 C SER 93 10.661 20.935 16.225 1.00 62.49
    ATOM 714 O SER 93 10.719 22.094 15.811 1.00 63.13
    ATOM 715 N ALA 94 11.724 20.281 16.677 1.00 63.76
    ATOM 716 CA ALA 94 13.019 20.930 16.716 1.00 65.86
    ATOM 717 CB ALA 94 12.971 22.123 17.669 1.00 65.68
    ATOM 718 C ALA 94 14.139 19.988 17.127 1.00 67.27
    ATOM 719 O ALA 94 14.181 19.508 18.260 1.00 67.78
    ATOM 720 N GLY 95 15.048 19.727 16.198 1.00 68.46
    ATOM 721 CA GLY 95 16.180 18.872 16.502 1.00 69.61
    ATOM 722 C GLY 95 15.883 17.489 17.045 1.00 69.64
    ATOM 723 O GLY 95 15.826 17.281 18.257 1.00 70.06
    ATOM 724 N SER 96 15.692 16.542 16.134 1.00 68.83
    ATOM 725 CA SER 96 15.434 15.151 16.487 1.00 68.81
    ATOM 726 CB SER 96 16.734 14.514 16.984 1.00 67.99
    ATOM 727 CG SER 96 17.708 14.490 15.954 1.00 69.91
    ATOM 728 C SER 96 14.320 14.901 17.514 1.00 68.02
    ATOM 729 O SER 96 13.902 13.759 17.726 1.00 69.37
    ATOM 730 N ASP 97 13.819 15.959 18.135 1.00 65.66
    ATOM 731 CA ASP 97 12.792 15.801 19.149 1.00 61.46
    ATOM 732 CB ASP 97 13.427 16.128 20.508 1.00 64.43
    ATOM 733 CC ASP 97 12.454 16.730 21.498 1.00 68.00
    ATOM 734 OD1 ASP 97 11.686 17.632 21.096 1.00 70.75
    ATOM 735 OD2 ASP 97 12.480 16.339 22.690 1.00 69.61
    ATOM 736 C ASP 97 11.563 16.657 18.874 1.00 57.75
    ATOM 737 O ASP 97 11.662 17.760 18.342 1.00 56.47
    ATOM 738 N ILE 98 10.399 16.116 19.220 1.00 54.15
    ATOM 739 CA ILE 98 9.129 16.810 19.063 1.00 48.95
    ATOM 740 CS ILE 98 8.069 15.928 18.372 1.00 48.45
    ATOM 741 CG2 ILE 98 6.724 16.639 18.364 1.00 46.97
    ATOM 742 CG1 ILE 98 8.519 15.571 16.955 1.00 47.46
    ATOM 743 CD1 ILE 98 7.566 14.651 16.22 91.00 45.26
    ATOM 744 C ILE 98 8.633 17.073 20.469 1.00 48.06
    ATOM 745 O ILE 98 8.669 16.187 21.310 1.00 47.48
    ATOM 746 N ARG 99 8.202 18.291 20.752 1.00 46.99
    ATOM 747 CA ARG 99 7.660 18.556 22.069 1.00 47.18
    ATOM 748 CB ARG 99 8.347 19.738 22.730 1.00 48.61
    ATOM 749 CC ARG 99 9.780 19.436 23.088 1.00 52.92
    ATOM 750 CD ARG 99 10.435 20.597 23.774 1.00 53.16
    ATOM 751 NE ARG 99 11.875 20.550 23.580 1.00 59.38
    ATOM 752 CZ ARG 99 12.656 19.533 23.930 1.00 62.25
    ATOM 753 NH1 ARG 99 12.137 18.453 24.507 1.00 63.64
    ATOM 754 NH2 ARG 99 13.963 19.594 23.687 1.00 64.01
    ATOM 755 C ARG 99 6.182 18.816 21.892 1.00 45.57
    ATOM 756 O ARG 99 5.748 19.325 20.853 1.00 45.31
    ATOM 757 N VAL 100 5.408 18.451 22.901 1.00 43.60
    ATOM 758 CA VAL 100 3.967 18.601 22.834 1.00 42.41
    ATOM 759 CB VAL 100 3.295 17.210 22.698 1.00 42.94
    ATOM 760 CG1 VAL 100 1.774 17.350 22.702 1.00 44.36
    ATOM 761 CG2 VAL 100 3.792 16.508 21.427 1.00 41.91
    ATOM 762 C VAL 100 3.417 19.310 24.065 1.00 41.58
    ATOM 763 O VAL 100 3.209 18.698 25.110 1.00 40.47
    ATOM 764 N PRO 101 3.236 20.632 23.977 1.00 41.65
    ATOM 765 CD PRO 101 3.568 21.570 22.889 1.00 41.07
    ATOM 766 CA PRO 101 2.696 21.361 25.122 1.00 39.69
    ATOM 767 CB PRO 101 2.830 22.809 24.675 1.00 40.75
    ATOM 768 CO PRO 101 2.608 22.690 23.169 1.00 40.97
    ATOM 769 C PRO 101 1.243 20.906 25.263 1.00 38.36
    ATOM 770 O PRO 101 0.566 20.638 24.273 1.00 36.38
    ATOM 771 N LEU 102 0.766 20.816 26.491 1.00 37.40
    ATOM 772 CA LEU 102 −0.597 20.349 26.728 1.00 36.57
    ATOM 773 CS LEU 102 −0.536 19.081 27.605 1.00 36.65
    ATOM 774 CG LEU 102 0.247 17.915 26.968 1.00 34.92
    ATOM 775 CD1 LEU 102 0.509 16.837 27.969 1.00 34.58
    ATOM 776 CD2 LEU 102 −0.518 17.391 25.785 1.00 31.23
    ATOM 777 C LEU 102 −1.536 21.375 27.364 1.00 35.46
    ATOM 778 O LEU 102 −1.148 22.129 28.247 1.00 32.96
    ATOM 779 N PRO 103 −2.809 21.379 26.945 1.00 37.48
    ATOM 780 CD PRO 103 −3.456 20.510 25.948 1.00 38.55
    ATOM 781 CA PRO 103 −3.797 22.311 27.485 1.00 39.19
    ATOM 782 CS PRO 103 −5.015 22.043 26.599 1.00 38.49
    ATOM 783 CC PRO 103 −4.921 20.592 26.384 1.00 36.60
    ATOM 784 C PRO 103 −4.055 21.999 28.960 1.00 41.74
    ATOM 785 O PRO 103 −3.955 20.850 29.388 1.00 41.12
    ATOM 786 N PRO 104 −4.386 23.018 29.763 1.00 45.20
    ATOM 787 CD PRO 104 −4.533 24.458 29.473 1.00 46.97
    ATOM 788 CA PRO 104 −4.643 22.772 31.184 1.00 44.68
    ATOM 789 CS PRO 104 −4.783 24.177 31.745 1.00 44.21
    ATOM 790 CC PRO 104 −5.461 24.893 30.601 1.00 47.15
    ATOM 791 C PRO 104 −5.911 21.957 31.390 1.00 45.36
    ATOM 792 O PRO 104 −6.735 21.812 30.480 1.00 46.09
    ATOM 793 N LEU 105 −6.056 21.400 32.580 1.00 44.16
    ATOM 794 CA LEU 105 −7.260 20.654 32.889 1.00 43.98
    ATOM 795 CE LEU 105 −7.208 20.155 34.327 1.00 42.52
    ATOM 796 CC LEU 105 −6.071 19.208 34.668 1.00 41.22
    ATOM 797 CD1 LEU 105 −6.052 18.923 36.174 1.00 40.33
    ATOM 798 CD2 LEU 105 −6.263 17.944 33.861 1.00 39.80
    ATOM 799 C LEU 105 −8.398 21.655 32.739 1.00 43.38
    ATOM 800 O LEU 105 −8.199 22.860 32.924 1.00 44.06
    ATOM 801 N THR 106 −9.588 21.175 32.412 1.00 41.61
    ATOM 802 CA THR 106 −10.709 22.087 32.255 1.00 42.62
    ATOM 803 CE THR 106 −11.943 21.367 31.673 1.00 40.92
    ATOM 804 OG1 THR 106 −12.315 20.274 32.518 1.00 38.13
    ATOM 805 OG2 THR 106 −11.626 20.847 30.278 1.00 41.50
    ATOM 806 C THR 106 −11.095 22.774 33.566 1.00 43.42
    ATOM 807 O THR 106 −10.777 22.301 34.651 1.00 40.88
    ATOM 808 N GLU 107 −11.777 23.903 33.439 1.00 46.29
    ATOM 809 CA GLU 107 −12.225 24.682 34.578 1.00 50.04
    ATOM 810 CE GLU 107 −13.100 25.840 34.094 1.00 55.08
    ATOM 811 CG GLU 107 −12.939 27.144 34.864 1.00 61.74
    ATOM 812 CO GLU 107 −11.783 27.997 34.337 1.00 65.24
    ATOM 813 OE1 GLU 107 −11.844 28.413 33.158 1.00 66.53
    ATOM 814 OE2 GLU 107 −10.820 28.257 35.096 1.00 68.36
    ATOM 815 C GLU 107 −13.064 23.760 35.464 1.00 49.91
    ATOM 816 O GLU 107 −12.860 23.673 36.677 1.00 49.39
    ATOM 817 N GLU 108 −14.004 23.067 34.835 1.00 49.83
    ATOM 818 CA GLU 108 −14.894 22.146 35.526 1.00 50.79
    ATOM 819 CE GLU 108 −15.887 21.532 34.523 1.00 52.77
    ATOM 820 CG GLU 108 −17.030 20.740 35.160 1.00 57.30
    ATOM 821 CD GLU 108 −17.865 21.574 36.143 1.00 60.57
    ATOM 822 OE1 GLU 108 −18.370 22.655 35.757 1.00 61.21
    ATOM 823 OE2 GLU 108 −18.018 21.141 37.310 1.00 63.46
    ATOM 824 C GLU 108 −14.126 21.043 36.262 1.00 49.48
    ATOM 825 O GLU 108 −14.509 20.636 37.369 1.00 48.71
    ATOM 826 N ARG 109 −13.042 20.566 35.656 1.00 47.01
    ATOM 827 CA ARG 109 −12.224 19.528 36.278 1.00 45.40
    ATOM 828 CE ARG 109 −11.211 18.982 35.270 1.00 45.36
    ATOM 829 CG ARG 109 −10.308 17.876 35.792 1.00 46.17
    ATOM 830 CD ARG 109 −11.096 16.681 36.378 1.00 48.74
    ATOM 831 NE ARG 109 −10.190 15.611 36.810 1.00 51.07
    ATOM 832 CZ ARG 109 −10.550 14.537 37.515 1.00 50.91
    ATOM 833 NH1 ARG 109 −11.810 14.363 37.886 1.00 50.39
    ATOM 834 NH2 ARG 109 −9.640 13.643 37.868 1.00 51.02
    ATOM 835 C ARG 109 −11.497 20.076 37.508 1.00 44.07
    ATOM 836 O ARG 109 −11.536 19.484 38.582 1.00 41.18
    ATOM 837 N ARG 110 −10.836 21.214 37.357 1.00 44.70
    ATOM 838 CA ARG 110 −10.128 21.790 38.484 1.00 45.62
    ATOM 839 CE ARG 110 −9.532 23.145 38.109 1.00 49.45
    ATOM 840 CC ARG 110 −8.769 23.823 39.239 1.00 55.43
    ATOM 841 CD ARG 110 −7.813 24.933 38.761 1.00 58.14
    ATOM 842 NE ARG 110 −8.383 25.798 37.729 1.00 59.04
    ATOM 843 CZ ARG 110 −8.436 25.487 36.435 1.00 61.75
    ATOM 844 NH1 ARG 110 −7.945 24.328 36.006 1.00 61.67
    ATOM 845 NH2 ARG 110 −8.993 26.331 35.572 1.00 61.81
    ATOM 846 C ARG 110 −11.072 21.920 39.667 1.00 45.36
    ATOM 847 O ARG 110 −10.697 21.602 40.804 1.00 44.82
    ATOM 848 N LYS 111 −12.300 22.363 39.404 1.00 44.64
    ATOM 849 CA LYS 111 −13.283 22.500 40.476 1.00 46.68
    ATOM 850 CE LYS 111 −14.549 23.225 39.997 1.00 48.26
    ATOM 851 CG LYS 111 −14.313 24.695 39.665 1.00 53.39
    ATOM 852 CD LYS 111 −15.621 25.451 39.447 1.00 56.17
    ATOM 853 CE LYS 111 −15.382 26.917 39.046 1.00 58.69
    ATOM 854 NE LYS 111 −14.714 27.105 37.709 1.00 56.88
    ATOM 855 C LYS 111 −13.640 21.138 41.067 1.00 44.82
    ATOM 856 O LYS 111 −13.726 21.010 42.276 1.00 44.23
    ATOM 857 N ASP 112 −13.841 20.125 40.231 1.00 45.41
    ATOM 858 CA ASP 112 −14.141 18.793 40.762 1.00 47.37
    ATOM 859 CB ASP 112 −14.362 17.766 39.634 1.00 49.39
    ATOM 860 CG ASP 112 −15.388 18.222 38.599 1.00 56.37
    ATOM 861 OD1 ASP 112 −16.549 18.548 38.976 1.00 58.22
    ATOM 862 OD2 ASP 112 −15.030 18.245 37.388 1.00 58.89
    ATOM 863 C ASP 112 −12.966 18.304 41.657 1.00 46.29
    ATOM 864 O ASP 112 −13.179 17.652 42.672 1.00 47.08
    ATOM 865 N LEU 113 −11.728 18.622 41.288 1.00 42.72
    ATOM 866 CA LEU 113 −10.582 18.181 42.084 1.00 40.11
    ATOM 867 CB LEU 113 −9.323 18.172 41.222 1.00 37.15
    ATOM 868 CG LEU 113 −9.390 17.125 40.111 1.00 36.21
    ATOM 869 CD1 LEU 113 −8.274 17.320 39.106 1.00 36.61
    ATOM 870 CD2 LEU 113 −9.341 15.744 40.742 1.00 37.64
    ATOM 871 C LEU 113 −10.370 19.027 43.340 1.00 39.82
    ATOM 872 O LEU 113 −9.973 18.509 44.400 1.00 37.85
    ATOM 873 N THR 114 −10.638 20.325 43.228 1.00 38.24
    ATOM 874 CA THR 114 −10.479 21.206 44.375 1.00 37.09
    ATOM 875 CB THR 114 −10.828 22.666 44.019 1.00 36.57
    ATOM 876 OG1 THR 114 −9.924 23.153 43.017 1.00 33.29
    ATOM 877 OG2 THR 114 −10.723 23.547 45.252 1.00 37.73
    ATOM 878 C THR 114 −11.459 20.708 45.417 1.00 39.56
    ATOM 879 O THR 114 −11.163 20.652 46.611 1.00 37.29
    ATOM 880 N LYS 115 −12.638 20.319 44.946 1.00 40.54
    ATOM 881 CA LYS 115 −13.661 19.842 45.848 1.00 41.37
    ATOM 882 CB LYS 115 −14.974 19.666 45.083 1.00 45.84
    ATOM 883 CC LYS 115 −16.101 19.059 45.880 1.00 49.09
    ATOM 884 CD LYS 115 −17.418 19.248 45.151 1.00 53.42
    ATOM 885 CE LYS 115 −18.522 18.381 45.760 1.00 56.03
    ATOM 886 NE LYS 115 −18.314 16.917 45.456 1.00 57.60
    ATOM 887 C LYS 115 −13.242 18.561 46.542 1.00 38.50
    ATOM 888 O LYS 115 −13.495 18.382 47.720 1.00 37.67
    ATOM 889 N ILE 116 −12.560 17.685 45.823 1.00 38.50
    ATOM 890 CA ILE 116 −12.133 16.434 46.419 1.00 37.98
    ATOM 891 CB ILE 116 −11.617 15.452 45.374 1.00 39.26
    ATOM 892 CG2 ILE 116 −11.367 14.104 46.027 1.00 39.71
    ATOM 893 CG1 ILE 116 −12.640 15.297 44.257 1.00 39.44
    ATOM 894 CD1 ILE 116 −13.980 14.876 44.742 1.00 42.71
    ATOM 895 C ILE 116 −11.050 16.602 47.474 1.00 37.87
    ATOM 896 O ILE 116 −11.127 15.979 48.537 1.00 38.93
    ATOM 897 N VAL 117 −10.041 17.425 47.209 1.00 35.17
    ATOM 898 CA VAL 117 −8.996 17.584 48.220 1.00 35.04
    ATOM 899 CB VAL 117 −7.764 18.330 47.666 1.00 32.48
    ATOM 900 CG1 VAL 117 −7.220 17.566 46.481 1.00 33.10
    ATOM 901 CG2 VAL 117 −8.123 19.714 47.248 1.00 32.62
    ATOM 902 C VAL 117 −9.560 18.268 49.468 1.00 33.43
    ATOM 903 O VAL 117 −9.215 17.897 50.596 1.00 33.84
    ATOM 904 N ARG 118 −10.442 19.246 49.274 1.00 33.17
    ATOM 905 CA ARG 118 −11.075 19.918 50.419 1.00 33.39
    ATOM 906 CB ARG 118 −11.982 21.060 49.961 1.00 29.92
    ATOM 907 CC ARG 118 −11.192 22.288 49.649 1.00 32.89
    ATOM 908 CD ARG 118 −12.029 23.334 49.036 1.00 37.88
    ATOM 909 NE ARG 118 −11.219 24.462 48.622 1.00 41.77
    ATOM 910 CZ ARG 118 −11.695 25.532 48.003 1.00 46.19
    ATOM 911 NH1 ARG 118 −12.987 25.626 47.718 1.00 47.00
    ATOM 912 NH2 ARG 118 −10.871 26.517 47.679 1.00 49.71
    ATOM 913 C ARG 118 −11.859 18.945 51.297 1.00 32.57
    ATOM 914 O ARG 118 −11.806 19.046 52.515 1.00 36.89
    ATOM 915 N GLY 119 −12.570 18.008 50.677 1.00 30.46
    ATOM 916 CA GLY 119 −13.322 17.018 51.423 1.00 29.23
    ATOM 917 C GLY 119 −12.407 16.082 52.202 1.00 29.62
    ATOM 918 O GLY 119 −12.702 15.722 53.335 1.00 30.06
    ATOM 919 N GLU 120 −11.294 15.692 51.593 1.00 30.52
    ATOM 920 CA GLU 120 −10.321 14.827 52.241 1.00 31.96
    ATOM 921 CB GLU 120 −9.281 14.349 51.222 1.00 34.72
    ATOM 922 CC GLU 120 −9.912 13.483 50.152 1.00 39.64
    ATOM 923 CD GLU 120 −8.962 13.047 49.049 1.00 42.12
    ATOM 924 OE1 GLU 120 −9.414 12.270 48.184 1.00 44.99
    ATOM 925 OE2 GLU 120 −7.780 13.461 49.030 1.00 43.53
    ATOM 926 C GLU 120 −9.649 15.592 53.368 1.00 32.43
    ATOM 927 O GLU 120 −9.499 15.063 54.462 1.00 35.07
    ATOM 928 N ALA 121 −9.260 16.845 53.124 1.00 32.32
    ATOM 929 CA ALA 121 −8.631 17.616 54.186 1.00 31.62
    ATOM 930 CB ALA 121 −8.336 19.035 53.720 1.00 28.12
    ATOM 931 C ALA 121 −9.624 17.634 55.344 1.00 34.83
    ATOM 932 O ALA 121 −9.293 17.273 56.475 1.00 36.01
    ATOM 933 N GLU 122 −10.858 18.018 55.026 1.00 35.31
    ATOM 934 CA GLU 122 −11.944 18.115 55.992 1.00 36.50
    ATOM 935 CB GLU 122 −13.259 18.348 55.242 1.00 39.77
    ATOM 936 CC GLU 122 −14.201 19.288 55.924 1.00 39.83
    ATOM 937 CD GLU 122 −14.446 18.903 57.343 1.00 40.58
    ATOM 938 OE1 GLU 122 −14.903 17.768 57.553 1.00 44.91
    ATOM 939 OE2 GLU 122 −14.184 19.728 58.250 1.00 41.71
    ATOM 940 C GLU 122 −12.028 16.836 56.808 1.00 36.15
    ATOM 941 O GLU 122 −12.081 16.875 58.037 1.00 34.04
    ATOM 942 N GLN 123 −12.053 15.698 56.115 1.00 36.64
    ATOM 943 CA GLN 123 −12.099 14.413 56.791 1.00 38.70
    ATOM 944 CB GLN 123 −12.070 13.295 55.751 1.00 40.64
    ATOM 945 CC GLN 123 −11.999 11.888 56.331 1.00 45.87
    ATOM 946 CD GLN 123 −10.575 11.398 56.525 1.00 50.88
    ATOM 947 OE1 GLN 123 −9.774 11.378 55.579 1.00 54.39
    ATOM 948 NE2 GLN 123 −10.254 10.983 57.739 1.00 50.01
    ATOM 949 C GLN 123 −10.910 14.277 57.760 1.00 37.35
    ATOM 950 O GLN 123 −11.070 13.855 58.900 1.00 37.65
    ATOM 951 N ALA 124 −9.721 14.652 57.296 1.00 35.43
    ATOM 952 CA ALA 124 −8.522 14.576 58.123 1.00 34.84
    ATOM 953 CB ALA 124 −7.252 15.024 57.307 1.00 33.06
    ATOM 954 C ALA 124 −8.670 15.431 59.384 1.00 32.69
    ATOM 955 O ALA 124 −8.236 15.024 60.470 1.00 31.59
    ATOM 956 N ARG 125 −9.266 16.612 59.237 1.00 29.13
    ATOM 957 CA ARG 125 −9.464 17.488 60.377 1.00 30.40
    ATOM 958 CB ARG 125 −9.883 18.901 59.943 1.00 33.05
    ATOM 959 CC ARG 125 −8.734 19.821 59.545 1.00 36.01
    ATOM 960 CD ARG 125 −9.244 21.249 59.398 1.00 37.50
    ATOM 961 NE ARG 125 −10.102 21.309 58.240 1.00 41.70
    ATOM 962 CZ ARG 125 −9.683 21.653 57.037 1.00 39.98
    ATOM 963 NE1 ARG 125 −8.415 21.979 56.856 1.00 42.73
    ATOM 964 NH2 ARG 125 −10.502 21.568 56.007 1.00 37.33
    ATOM 965 C ARG 125 −10.468 16.943 61.386 1.00 30.54
    ATOM 966 O ARG 125 −10.296 17.144 62.580 1.00 31.78
    ATOM 967 N VAL 126 −11.511 16.262 60.923 1.00 30.13
    ATOM 968 CA VAL 126 −12.474 15.683 61.847 1.00 29.18
    ATOM 969 CB VAL 126 −13.762 15.137 61.107 1.00 30.93
    ATOM 970 CG1 VAL 126 −14.721 14.517 62.104 1.00 26.39
    ATOM 971 CG2 VAL 126 −14.509 16.292 60.354 1.00 28.70
    ATOM 972 C VAL 126 −11.754 14.531 62.583 1.00 30.04
    ATOM 973 O VAL 126 −11.820 14.422 63.816 1.00 31.66
    ATOM 974 N ALA 127 −11.031 13.698 61.849 1.00 28.96
    ATOM 975 CA ALA 127 −10.337 12.583 62.492 1.00 30.05
    ATOM 976 CB ALA 127 −9.574 11.753 61.434 1.00 29.30
    ATOM 977 C ALA 127 −9.374 13.067 63.589 1.00 29.90
    ATOM 978 O ALA 127 −9.308 12.493 64.673 1.00 31.73
    ATOM 979 N VAL 128 −8.643 14.137 63.312 1.00 31.82
    ATOM 980 CA VAL 128 −7.686 14.694 64.264 1.00 31.72
    ATOM 981 CB VAL 128 −6.844 15.819 63.581 1.00 33.25
    ATOM 982 CG1 VAL 128 −5.997 16.559 64.626 1.00 35.15
    ATOM 983 CG2 VAL 128 −5.936 15.195 62.504 1.00 28.98
    ATOM 984 C VAL 128 −8.346 15.229 65.546 1.00 34.61
    ATOM 985 O VAL 128 −7.795 15.063 66.644 1.00 34.11
    ATOM 986 N ARG 129 −9.514 15.863 65.396 1.00 34.97
    ATOM 987 CA ARG 129 −10.285 16.415 66.518 1.00 36.61
    ATOM 988 CB ARG 129 −11.416 17.335 66.048 1.00 39.01
    ATOM 989 CG ARG 129 −11.084 18.404 65.030 1.00 42.15
    ATOM 990 CD ARG 129 −12.401 18.970 64.452 1.00 43.95
    ATOM 991 NE ARG 129 −12.193 19.869 63.321 1.00 46.07
    ATOM 992 CZ ARG 129 −11.349 20.900 63.338 1.00 47.32
    ATOM 993 NH1 ARG 129 −10.623 21.162 64.426 1.00 47.90
    ATOM 994 NH2 ARG 129 −11.245 21.682 62.284 1.00 44.26
    ATOM 995 C ARG 129 −10.964 15.279 67.291 1.00 36.52
    ATOM 996 O ARG 129 −11.378 15.471 68.421 1.00 37.01
    ATOM 997 N ASN 130 −11.146 14.122 66.664 1.00 35.22
    ATOM 998 CA ASN 130 −11.762 13.006 67.376 1.00 34.87
    ATOM 999 CB ASN 130 −12.340 11.970 66.367 1.00 33.94
    ATOM 1000 CC ASN 130 −13.708 12.406 65.788 1.00 34.78
    ATOM 1001 OD1 ASN 130 −14.138 11.953 64.723 1.00 32.62
    ATOM 1002 ND2 ASN 130 −14.386 13.282 66.507 1.00 29.90
    ATOM 1003 C ASN 130 −10.679 12.394 68.285 1.00 36.22
    ATOM 1004 O ASN 130 −10.954 11.958 69.403 1.00 35.32
    ATOM 1005 N VAL 131 −9.439 12.378 67.803 1.00 37.20
    ATOM 1006 CA VAL 131 −8.359 11.849 68.607 1.00 38.51
    ATOM 1007 CB VAL 131 −7.044 11.745 67.811 1.00 38.07
    ATOM 1008 CG1 VAL 131 −5.916 11.279 68.733 1.00 38.68
    ATOM 1009 CG2 VAL 131 −7.215 10.797 66.651 1.00 37.75
    ATOM 1010 C VAL 131 −8.148 12.816 69.782 1.00 40.98
    ATOM 1011 O VAL 131 −8.009 12.364 70.930 1.00 40.00
    ATOM 1012 N ARG 132 −8.141 14.130 69.499 1.00 40.45
    ATOM 1013 CA ARG 132 −7.943 15.133 70.547 1.00 42.82
    ATOM 1014 CB ARG 132 −8.178 16.559 70.046 1.00 42.45
    ATOM 1015 CC ARG 132 −8.001 17.603 71.165 1.00 48.52
    ATOM 1016 CD ARG 132 −8.630 19.001 70.918 1.00 50.87
    ATOM 1017 NE ARG 132 −10.080 18.961 71.107 1.00 57.79
    ATOM 1018 CZ ARG 132 −10.961 18.537 70.198 1.00 59.32
    ATOM 1019 NH1 ARG 132 −10.562 18.114 69.011 1.00 62.98
    ATOM 1020 NH2 ARG 132 −12.246 18.467 70.501 1.00 59.20
    ATOM 1021 C ARG 132 −8.935 14.855 71.658 1.00 44.60
    ATOM 1022 O ARG 132 −8.572 14.723 72.828 1.00 42.61
    ATOM 1023 N ARG 133 −10.196 14.767 71.258 1.00 46.77
    ATOM 1024 CA ARG 133 −11.300 14.497 72.162 1.00 50.55
    ATOM 1025 CB ARG 133 −12.582 14.317 71.351 1.00 52.21
    ATOM 1026 CC ARG 133 −13.848 14.227 72.170 1.00 57.48
    ATOM 1027 CD ARG 133 −15.000 13.921 71.244 1.00 63.69
    ATOM 1028 NE ARG 133 −14.973 14.767 70.047 1.00 68.51
    ATOM 1029 CE ARG 133 −15.071 16.097 70.051 1.00 70.24
    ATOM 1030 NH1 ARG 133 −15.206 16.754 71.194 1.00 72.00
    ATOM 1031 NH2 ARG 133 −15.030 16.772 68.908 1.00 70.13
    ATOM 1032 C ARG 133 −11.035 13.233 72.985 1.00 52.21
    ATOM 1033 O ARG 133 −11.082 13.265 74.218 1.00 51.81
    ATOM 1034 N ASP 134 −10.763 12.123 72.302 1.00 52.59
    ATOM 1035 CA ASP 134 −10.505 10.868 72.989 1.00 54.66
    ATOM 1036 CB ASP 134 −10.116 9.772 72.003 1.00 56.25
    ATOM 1037 CG ASP 134 −9.959 8.427 72.682 1.00 59.16
    ATOM 1038 OD1 ASP 134 −10.953 7.972 73.290 1.00 60.25
    ATOM 1039 OD2 ASP 134 −8.860 7.830 72.622 1.00 58.00
    ATOM 1040 C ASP 134 −9.404 11.007 74.028 1.00 55.18
    ATOM 1041 O ASP 134 −9.400 10.306 75.036 1.00 54.08
    ATOM 1042 N ALA 135 −8.454 11.895 73.768 1.00 56.31
    ATOM 1043 CA ALA 135 −7.369 12.133 74.714 1.00 57.14
    ATOM 1044 CB ALA 135 −6.267 12.944 74.057 1.00 55.72
    ATOM 1045 C ALA 135 −7.954 12.902 75.896 1.00 57.58
    ATOM 1046 O ALA 135 −7.872 12.462 77.040 1.00 57.83
    ATOM 1047 N ASN 136 −8.549 14.053 75.599 1.00 59.12
    ATOM 1048 CA ASN 136 −9.176 14.900 76.610 1.00 61.05
    ATOM 1049 CB ASN 136 −10.100 15.940 75.955 1.00 60.54
    ATOM 1050 CG ASN 136 −9.392 16.808 74.912 1.00 62.07
    ATOM 1051 OD1 ASN 136 −8.439 17.531 75.220 1.00 62.74
    ATOM 1052 ND2 ASN 136 −9.870 16.748 73.673 1.00 61.09
    ATOM 1053 C ASN 136 −10.007 14.036 77.565 1.00 62.37
    ATOM 1054 O ASN 136 −9.965 14.233 78.773 1.00 63.28
    ATOM 1055 N ASP 137 −10.758 13.084 77.013 1.00 64.25
    ATOM 1056 CA ASP 137 −11.601 12.191 77.812 1.00 66.76
    ATOM 1057 CB ASP 137 −12.272 11.131 76.940 1.00 67.37
    ATOM 1058 CG ASP 137 −13.250 11.714 75.967 1.00 68.71
    ATOM 1059 OD1 ASP 137 −14.119 12.507 76.398 1.00 68.32
    ATOM 1060 OD2 ASP 137 −13.156 11.359 74.772 1.00 70.08
    ATOM 1061 C ASP 137 −10.821 11.462 78.889 1.00 67.84
    ATOM 1062 O ASP 137 −11.079 11.616 80.081 1.00 68.36
    ATOM 1063 N LYS 138 −9.887 10.634 78.449 1.00 68.15
    ATOM 1064 CA LYS 138 −9.060 9.875 79.362 1.00 70.26
    ATOM 1065 CD LYS 138 −7.922 9.208 78.583 1.00 68.44
    ATOM 1066 CC LYS 138 −8.419 8.213 77.552 1.00 67.37
    ATOM 1067 CD LYS 138 −7.279 7.532 76.846 1.00 66.77
    ATOM 1068 CE LYS 138 −7.794 6.509 75.855 1.00 65.32
    ATOM 1069 NZ LYS 138 −6.661 5.798 75.206 1.00 65.96
    ATOM 1070 C LYS 138 −8.509 10.791 80.454 1.00 72.20
    ATOM 1071 O LYS 138 −8.637 10.492 81.642 1.00 71.61
    ATOM 1072 N VAL 139 −7.919 11.910 80.037 1.00 73.71
    ATOM 1073 CA VAL 139 −7.341 12.885 80.955 1.00 75.93
    ATOM 1074 CB VAL 139 −6.746 14.094 80.181 1.00 75.79
    ATOM 1075 CG1 VAL 139 −6.124 15.085 81.160 1.00 75.22
    ATOM 1076 CG2 VAL 139 −5.708 13.615 79.168 1.00 74.05
    ATOM 1077 C VAL 139 −8.357 13.416 81.974 1.00 77.78
    ATOM 1078 O VAL 139 −8.168 13.284 83.184 1.00 77.15
    ATOM 1079 N LYS 140 −9.432 14.017 81.471 1.00 80.32
    ATOM 1080 CA LYS 140 −10.486 14.591 82.309 1.00 82.58
    ATOM 1081 CD LYS 140 −11.531 15.266 81.413 1.00 83.61
    ATOM 1082 CG LYS 140 −12.436 16.253 82.117 1.00 84.66
    ATOM 1083 CD LYS 140 −11.616 17.410 82.677 1.00 86.11
    ATOM 1084 CE LYS 140 −12.504 18.499 83.265 1.00 87.30
    ATOM 1085 NZ LYS 140 −13.401 18.007 84.343 1.00 87.20
    ATOM 1086 C LYS 140 −11.161 13.524 83.185 1.00 83.58
    ATOM 1087 O LYS 140 −11.543 13.788 84.329 1.00 82.45
    ATOM 1088 N ALA 141 −11.297 12.321 82.633 1.00 85.31
    ATOM 1089 CA ALA 141 −11.917 11.201 83.331 1.00 87.36
    ATOM 1090 CE ALA 141 −12.222 10.079 82.349 1.00 87.57
    ATOM 1091 C ALA 141 −11.022 10.676 84.442 1.00 88.79
    ATOM 1092 O ALA 141 −11.479 10.439 85.555 1.00 89.29
    ATOM 1093 N LEU 142 −9.745 10.492 84.130 1.00 90.16
    ATOM 1094 CA LEU 142 −8.781 9.982 85.098 1.00 91.17
    ATOM 1095 CB LEU 142 −7.390 9.942 84.467 1.00 90.19
    ATOM 1096 CC LEU 142 −6.450 8.865 84.999 1.00 90.65
    ATOM 1097 CD1 LEU 142 −7.081 7.503 84.763 1.00 90.25
    ATOM 1098 CD2 LEU 142 −5.108 8.947 84.292 1.00 90.32
    ATOM 1099 C LEU 142 −8.782 10.889 86.330 1.00 92.69
    ATOM 1100 O LEU 142 −8.453 10.463 87.439 1.00 92.75
    ATOM 1101 N LEU 143 −9.175 12.141 86.120 1.00 94.14
    ATOM 1102 CA LEU 143 −9.243 13.134 87.188 1.00 95.26
    ATOM 1103 CB LEU 143 −9.537 14.513 86.585 1.00 94.57
    ATOM 1104 CC LEU 143 −9.684 15.706 87.531 1.00 93.49
    ATOM 1105 CD1 LEU 143 −8.373 15.956 88.248 1.00 93.46
    ATOM 1106 CD2 LEU 143 −10.091 16.933 86.735 1.00 92.61
    ATOM 1107 C LEU 143 −10.324 12.789 88.212 1.00 96.21
    ATOM 1108 O LEU 143 −10.246 13.195 89.370 1.00 95.47
    ATOM 1109 N LYS 144 −11.331 12.036 87.778 1.00 98.51
    ATOM 1110 CA LYS 144 −12.441 11.662 88.649 1.00 100.82
    ATOM 1111 CB LYS 144 −13.506 10.862 87.880 1.00 100.58
    ATOM 1112 CG LYS 144 −13.063 9.460 87.477 1.00 101.19
    ATOM 1113 CD LYS 144 −14.161 8.663 86.800 1.00 101.64
    ATOM 1114 CE LYS 144 −13.669 7.259 86.448 1.00 102.30
    ATOM 1115 NZ LYS 144 −14.711 6.420 85.783 1.00 102.31
    ATOM 1116 C LYS 144 −12.002 10.847 89.854 1.00 102.32
    ATOM 1117 O LYS 144 −12.148 11.296 90.990 1.00 102.94
    ATOM 1118 N ASP 145 −11.459 9.657 89.601 1.00 103.95
    ATOM 1119 CA ASP 145 −11.042 8.758 90.672 1.00 105.61
    ATOM 1120 CB ASP 145 −11.670 7.364 90.449 1.00 105.33
    ATOM 1121 CG ASP 145 −13.200 7.379 90.493 1.00 105.16
    ATOM 1122 OD1 ASP 145 −13.778 7.740 91.545 1.00 104.28
    ATOM 1123 OD2 ASP 145 −13.827 7.028 89.472 1.00 104.93
    ATOM 1124 C ASP 145 −9.533 8.600 90.897 1.00 106.36
    ATOM 1125 O ASP 145 −9.071 8.609 92.040 1.00 106.84
    ATOM 1126 N LYS 146 −8.764 8.475 89.817 1.00 107.05
    ATOM 1127 CA LYS 146 −7.323 8.273 89.963 1.00 107.59
    ATOM 1128 CB LYS 146 −6.829 7.347 88.840 1.00 107.69
    ATOM 1129 CC LYS 146 −7.534 5.979 88.885 1.00 108.01
    ATOM 1130 CD LYS 146 −6.967 4.952 87.912 1.00 107.88
    ATOM 1131 CE LYS 146 −7.653 3.595 88.104 1.00 107.75
    ATOM 1132 NZ LYS 146 −7.039 2.522 87.273 1.00 107.65
    ATOM 1133 C LYS 146 −6.449 9.527 90.091 1.00 108.00
    ATOM 1134 O LYS 146 −6.909 10.654 89.905 1.00 108.57
    ATOM 1135 N GLU 147 −5.183 9.286 90.415 1.00 107.89
    ATOM 1136 CA GLU 147 −4.118 10.269 90.678 1.00 107.41
    ATOM 1137 CB GLU 147 −2.780 9.531 90.640 1.00 108.68
    ATOM 1138 CG GLU 147 −2.465 8.846 89.314 1.00 109.90
    ATOM 1139 CD GLU 147 −1.146 8.093 89.368 1.00 111.13
    ATOM 1140 OE1 GLU 147 −1.020 7.174 90.206 1.00 111.54
    ATOM 1141 OE2 GLU 147 −0.235 8.421 88.579 1.00 111.87
    ATOM 1142 C GLU 147 −3.868 11.665 90.067 1.00 106.09
    ATOM 1143 O GLU 147 −3.101 12.420 90.647 1.00 107.03
    ATOM 1144 N ILE 148 −4.448 12.040 88.936 1.00 103.57
    ATOM 1145 CA ILE 148 −4.121 13.373 63.414 1.00 101.47
    ATOM 1146 CB ILE 148 −4.278 13.430 86.892 1.00 101.45
    ATOM 1147 CG2 ILE 148 −3.702 14.724 86.364 1.00 101.52
    ATOM 1148 CG1 ILE 148 −3.553 12.249 86.245 1.00 101.43
    ATOM 1149 CD1 ILE 148 −4.200 10.917 86.516 1.00 101.07
    ATOM 1150 C ILE 148 −4.974 14.484 89.025 1.00 100.34
    ATOM 1151 O ILE 148 −6.083 14.222 89.495 1.00 100.42
    ATOM 1152 N SER 149 −4.463 15.717 89.021 1.00 98.53
    ATOM 1153 CA SER 149 −5.202 16.873 89.556 1.00 96.84
    ATOM 1154 CB SER 149 −4.599 17.320 90.882 1.00 97.16
    ATOM 1155 OG SER 149 −3.248 17.689 90.706 1.00 97.28
    ATOM 1156 C SER 149 −5.203 18.049 88.579 1.00 95.62
    ATOM 1157 O SER 149 −4.454 18.056 87.608 1.00 95.10
    ATOM 1158 N GLU 150 −6.040 19.048 88.860 1.00 94.27
    ATOM 1159 CA GLU 150 −6.215 20.244 88.028 1.00 92.89
    ATOM 1160 CB GLU 150 −6.809 21.357 88.891 1.00 92.94
    ATOM 1161 CC GLU 150 −8.218 21.047 89.354 1.00 93.02
    ATOM 1162 CD GLU 150 −8.668 21.948 90.477 1.00 93.68
    ATOM 1163 OE1 GLU 150 −8.638 23.186 90.303 1.00 94.33
    ATOM 1164 OE2 GLU 150 −9.053 21.411 91.537 1.00 93.85
    ATOM 1165 C GLU 150 −5.050 20.810 87.203 1.00 91.59
    ATOM 1166 O GLU 150 −5.191 20.999 85.998 1.00 91.47
    ATOM 1167 N ASP 151 −3.919 21.092 87.839 1.00 90.68
    ATOM 1168 CA ASP 151 −2.770 21.644 87.116 1.00 89.65
    ATOM 1169 CB ASP 151 −1.743 22.205 88.091 1.00 89.29
    ATOM 1170 CG ASP 151 −2.240 23.431 88.812 1.00 89.52
    ATOM 1171 OD1 ASP 151 −2.566 24.421 83.129 1.00 88.83
    ATOM 1172 OD2 ASP 151 −2.304 23.408 90.059 1.00 90.30
    ATOM 1173 C ASP 151 −2.080 20.661 86.178 1.00 88.62
    ATOM 1174 O ASP 151 −1.342 21.065 85.284 1.00 89.06
    ATOM 1175 N ASP 152 −2.304 19.373 86.394 1.00 87.65
    ATOM 1176 CA ASP 152 −1.707 18.363 85.535 1.00 87.45
    ATOM 1177 CB ASP 152 −1.480 17.072 86.324 1.00 89.17
    ATOM 1178 CG ASP 152 −0.664 16.052 85.552 1.00 91.93
    ATOM 1179 OD1 ASP 152 −1.036 15.721 84.403 1.00 93.10
    ATOM 1180 OD2 ASP 152 −0.351 15.577 86.102 1.00 93.68
    ATOM 1181 C ASP 152 −2.700 18.127 84.388 1.00 85.95
    ATOM 1182 O ASP 152 −2.324 17.698 83.295 1.00 85.41
    ATOM 1183 N ASP 153 −3.969 18.422 84.670 1.00 83.73
    ATOM 1184 CA ASP 153 −5.066 18.284 83.720 1.00 80.91
    ATOM 1185 CB ASP 153 −6.409 18.406 84.458 1.00 80.84
    ATOM 1186 CC ASP 153 −7.619 18.190 83.552 1.00 79.88
    ATOM 1187 OD1 ASP 153 −8.752 18.336 84.056 1.00 79.70
    ATOM 1188 OD2 ASP 153 −7.452 17.873 82.355 1.00 79.02
    ATOM 1189 C ASP 153 −4.907 19.431 82.740 1.00 80.07
    ATOM 1190 O ASP 153 −4.879 19.227 81.526 1.00 79.99
    ATOM 1191 N ARG 154 −4.790 20.639 83.284 1.00 77.88
    ATOM 1192 CA ARG 154 −4.618 21.833 82.471 1.00 76.70
    ATOM 1193 CB ARG 154 −4.318 23.040 83.370 1.00 78.40
    ATOM 1194 CC ARG 154 −5.457 23.397 84.310 1.00 81.12
    ATOM 1195 CO ARG 154 −5.182 24.639 85.155 1.00 82.69
    ATOM 1196 NE ARG 154 −6.336 24.979 85.989 1.00 84.80
    ATOM 1197 CZ ARG 154 −6.376 25.999 86.843 1.00 86.51
    ATOM 1198 NH1 ARG 154 −5.320 26.794 86.983 1.00 87.33
    ATOM 1199 NH2 ARG 154 −7.470 26.225 87.561 1.00 86.40
    ATOM 1200 C ARG 154 −3.498 21.658 81.442 1.00 74.53
    ATOM 1201 O ARG 154 −3.743 21.703 80.236 1.00 73.89
    ATOM 1202 N ARG 155 −2.274 21.463 81.935 1.00 72.04
    ATOM 1203 CA ARG 155 −1.091 21.279 81.096 1.00 68.48
    ATOM 1204 CB ARG 155 0.122 20.941 81.964 1.00 69.87
    ATOM 1205 CC ARG 155 0.724 22.109 82.723 1.00 71.53
    ATOM 1206 CD ARG 155 1.847 21.617 83.630 1.00 73.51
    ATOM 1207 NE ARG 155 2.733 22.696 84.054 1.00 74.79
    ATOM 1208 CZ ARG 155 3.518 23.385 83.228 1.00 75.80
    ATOM 1209 NH1 ARG 155 3.531 23.113 81.927 1.00 75.28
    ATOM 1210 NH2 ARG 155 4.299 24.347 83.702 1.00 76.38
    ATOM 1211 C ARG 155 −1.288 20.178 80.074 1.00 65.87
    ATOM 1212 O ARG 155 −1.017 20.366 78.895 1.00 65.37
    ATOM 1213 N SER 156 −1.745 19.022 80.542 1.00 63.16
    ATOM 1214 CA SER 156 −1.993 17.882 79.676 1.00 60.61
    ATOM 1215 CB SER 156 −2.509 16.702 80.490 1.00 58.93
    ATOM 1216 CG SER 156 −2.937 15.672 79.622 1.00 59.68
    ATOM 1217 C SER 156 −3.008 18.224 78.591 1.00 59.40
    ATOM 1218 O SER 156 −2.854 17.810 77.446 1.00 58.17
    ATOM 1219 N GLN 157 −4.046 18.969 78.965 1.00 58.38
    ATOM 1220 CA GLN 157 −5.077 19.364 78.021 1.00 58.88
    ATOM 1221 CB GLN 157 −6.259 20.012 78.745 1.00 61.05
    ATOM 1222 CC GLN 157 −7.047 19.058 79.649 1.00 64.28
    ATOM 1223 CD GLN 157 −7.648 17.870 78.894 1.00 66.97
    ATOM 1224 OE1 GLN 157 −6.932 17.108 78.228 1.00 67.14
    ATOM 1225 NE2 GLN 157 −8.965 17.702 79.006 1.00 67.00
    ATOM 1226 C GLN 157 −4.492 20.327 77.009 1.00 59.44
    ATOM 1227 O GLN 157 −4.795 20.247 75.818 1.00 60.58
    ATOM 1228 N ASP 158 −3.638 21.226 77.482 1.00 57.75
    ATOM 1229 CA ASP 158 −2.992 22.194 76.611 1.00 57.99
    ATOM 1230 CB ASP 158 −2.371 23.334 77.447 1.00 59.91
    ATOM 1231 CC ASP 158 −3.427 24.269 78.068 1.00 63.30
    ATOM 1232 OD1 ASP 158 −3.051 25.214 78.805 1.00 65.17
    ATOM 1233 OD2 ASP 158 −4.635 24.070 77.815 1.00 64.66
    ATOM 1234 C ASP 158 −1.925 21.543 75.703 1.00 56.75
    ATOM 1235 O ASP 158 −1.823 21.902 74.525 1.00 53.72
    ATOM 1236 N ASP 159 −1.140 20.600 76.241 1.00 55.82
    ATOM 1237 CA ASP 159 −0.113 19.925 75.441 1.00 54.58
    ATOM 1238 CB ASP 159 0.695 18.888 76.250 1.00 58.46
    ATOM 1239 CC ASP 159 1.498 19.499 77.402 1.00 62.06
    ATOM 1240 OD1 ASP 159 2.156 20.552 77.223 1.00 63.94
    ATOM 1241 OD2 ASP 159 1.500 18.891 78.497 1.00 65.22
    ATOM 1242 C ASP 159 −0.813 19.197 74.301 1.00 52.55
    ATOM 1243 O ASP 159 −0.320 19.187 73.174 1.00 52.06
    ATOM 1244 N VAL 160 −1.963 18.592 74.606 1.00 49.12
    ATOM 1245 CA VAL 160 −2.759 17.870 73.603 1.00 49.39
    ATOM 1246 CB VAL 160 −3.943 17.083 74.265 1.00 49.22
    ATOM 1247 CG1 VAL 160 −4.896 16.550 73.202 1.00 48.51
    ATOM 1248 CG2 VAL 160 −3.402 15.922 75.096 1.00 48.41
    ATOM 1249 C VAL 160 −3.330 18.831 72.548 1.00 47.61
    ATOM 1250 O VAL 160 −3.330 18.524 71.348 1.00 45.78
    ATOM 1251 N GLN 161 −3.820 19.982 73.008 1.00 46.87
    ATOM 1252 CA GLN 161 −4.385 21.007 72.121 1.00 47.61
    ATOM 1253 CB GLN 161 −4.829 22.234 72.930 1.00 49.79
    ATOM 1254 CC GLN 161 −5.289 23.402 72.059 1.00 48.02
    ATOM 1255 CD GLN 161 −6.388 22.993 71.113 1.00 49.19
    ATOM 1256 OE1 GLN 161 −7.441 22.526 71.540 1.00 49.82
    ATOM 1257 NE2 GLN 161 −6.150 23.156 69.813 1.00 50.57
    ATOM 1258 C GLN 161 −3.356 21.461 71.088 1.00 46.12
    ATOM 1259 O GLN 161 −3.680 21.734 69.931 1.00 45.79
    ATOM 1260 N LYS 162 −2.113 21.544 71.535 1.00 44.41
    ATOM 1261 CA LYS 162 −1.026 21.969 70.687 1.00 44.22
    ATOM 1262 CB LYS 162 0.173 22.336 71.572 1.00 46.80
    ATOM 1263 CG LYS 162 −0.151 23.474 72.548 1.00 48.45
    ATOM 1264 CD LYS 162 1.042 23.861 73.410 1.00 55.00
    ATOM 1265 CE LYS 162 0.782 25.121 74.240 1.00 55.12
    ATOM 1266 NZ LYS 162 −0.428 24.984 75.112 1.00 58.91
    ATOM 1267 C LYS 162 −0.696 20.868 69.681 1.00 42.73
    ATOM 1268 O LYS 162 −0.371 21.148 68.538 1.00 44.92
    ATOM 1269 N LEU 163 −0.784 19.613 70.110 1.00 41.72
    ATOM 1270 CA LEU 163 −0.545 18.492 69.213 1.00 38.98
    ATOM 1271 CB LEU 163 −0.664 17.170 69.962 1.00 36.52
    ATOM 1272 CC LEU 163 0.584 16.760 70.740 1.00 40.24
    ATOM 1273 CD1 LEU 163 0.308 15.468 71.517 1.00 39.87
    ATOM 1274 CD2 LEU 163 1.757 16.585 69.753 1.00 36.04
    ATOM 1275 C LEU 163 −1.584 18.550 68.102 1.00 37.77
    ATOM 1276 O LEU 163 −1.290 18.274 66.950 1.00 37.29
    ATOM 1277 N THR 164 −2.808 18.907 68.476 1.00 38.49
    ATOM 1278 CA THR 164 −3.914 19.038 67.538 1.00 37.80
    ATOM 1279 CB THR 164 −5.209 19.367 68.298 1.00 38.14
    ATOM 1280 OG1 THR 164 −5.405 18.386 69.321 1.00 39.84
    ATOM 1281 OG2 THR 164 −6.412 19.353 67.366 1.00 36.51
    ATOM 1282 C THR 164 −3.584 20.170 66.562 1.00 38.02
    ATOM 1283 O THR 164 −3.753 20.016 65.356 1.00 38.15
    ATOM 1284 N ASP 165 −3.097 21.290 67.100 1.00 38.34
    ATOM 1285 CA ASP 165 −2.709 22.463 66.307 1.00 42.07
    ATOM 1286 CB ASP 165 −2.118 23.573 67.205 1.00 45.79
    ATOM 1287 CC ASP 165 −3.139 24.181 68.158 1.00 49.06
    ATOM 1288 OD1 ASP 165 −2.716 24.949 69.057 1.00 51.38
    ATOM 1289 OD2 ASP 165 −4.355 23.905 68.005 1.00 48.41
    ATOM 1290 C ASP 165 −1.670 22.124 65.251 1.00 40.17
    ATOM 1291 O ASP 165 −1.809 22.489 64.093 1.00 39.87
    ATOM 1292 N ALA 166 −0.607 21.443 65.658 1.00 39.77
    ATOM 1293 CA ALA 166 0.430 21.079 64.699 1.00 38.11
    ATOM 1294 CB ALA 166 1.659 20.514 65.439 1.00 36.43
    ATOM 1295 C ALA 166 −0.096 20.067 63.663 1.00 35.40
    ATOM 1296 O ALA 166 0.291 20.104 62.492 1.00 36.48
    ATOM 1297 N ALA 167 −0.976 19.165 64.086 1.00 32.91
    ATOM 1298 CA ALA 167 −1.506 18.188 63.148 1.00 32.56
    ATOM 1299 CB ALA 167 −2.260 17.075 63.900 1.00 27.68
    ATOM 1300 C ALA 167 −2.434 18.925 62.167 1.00 33.32
    ATOM 1301 O ALA 167 −2.378 18.708 60.957 1.00 32.20
    ATOM 1302 N ILE 168 −3.276 19.815 62.690 1.00 33.15
    ATOM 1303 CA ILE 168 −4.173 20.549 61.817 1.00 34.96
    ATOM 1304 CB ILE 168 −5.224 21.422 62.650 1.00 36.24
    ATOM 1305 CG2 ILE 168 −5.269 22.847 62.144 1.00 33.31
    ATOM 1306 CG1 ILE 168 −6.660 20.870 62.468 1.00 38.04
    ATOM 1307 CD1 ILE 168 −6.924 19.486 62.974 1.00 35.50
    ATOM 1308 C ILE 168 −3.318 21.409 60.882 1.00 34.13
    ATOM 1309 O ILE 168 −3.619 21.546 59.707 1.00 32.89
    ATOM 1310 N LYS 169 −2.221 21.957 61.389 1.00 37.28
    ATOM 1311 CA LYS 169 −1.364 22.798 60.539 1.00 39.15
    ATOM 1312 CB LYS 169 −0.261 23.461 61.365 1.00 43.88
    ATOM 1313 CC LYS 169 0.749 24.246 60.509 1.00 48.23
    ATOM 1314 CD LYS 169 1.813 24.917 61.371 1.00 52.07
    ATOM 1315 CE LYS 169 2.902 25.630 60.530 1.00 54.04
    ATOM 1316 NZ LYS 169 2.387 26.724 59.637 1.00 55.30
    ATOM 1317 C LYS 169 −0.744 22.059 59.342 1.00 36.63
    ATOM 1318 O LYS 169 −0.649 22.616 58.258 1.00 36.33
    ATOM 1319 N LYS 170 −0.336 20.808 59.525 1.00 36.30
    ATOM 1320 CA LYS 170 0.248 20.042 58.412 1.00 35.20
    ATOM 1321 CB LYS 170 0.798 18.694 58.907 1.00 35.03
    ATOM 1322 CG LYS 170 2.026 18.786 59.780 1.00 35.34
    ATOM 1323 CD LYS 170 2.268 17.475 60.522 1.00 37.56
    ATOM 1324 CE LYS 170 3.459 17.597 61.468 1.00 43.01
    ATOM 1325 NZ LYS 170 3.540 16.490 62.471 1.00 43.46
    ATOM 1326 C LYS 170 −0.797 19.788 57.322 1.00 34.52
    ATOM 1327 O LYS 170 −0.503 19.889 56.140 1.00 36.16
    ATOM 1328 N ILE 171 −2.013 19.439 57.734 1.00 34.26
    ATOM 1329 CA ILE 171 −3.102 19.180 56.802 1.00 33.78
    ATOM 1330 CB ILE 171 −4.433 18.788 57.543 1.00 31.23
    ATOM 1331 CG2 ILE 171 −5.614 18.890 56.563 1.00 28.07
    ATOM 1332 CG1 ILE 171 −4.312 17.386 58.162 1.00 26.76
    ATOM 1333 CD1 ILE 171 −5.344 17.070 59.301 1.00 20.37
    ATOM 1334 C ILE 171 −3.340 20.430 55.977 1.00 32.17
    ATOM 1335 O ILE 171 −3.469 20.358 54.769 1.00 34.63
    ATOM 1336 N GLU 172 −3.379 21.580 56.629 1.00 34.69
    ATOM 1337 CA GLU 172 −3.606 22.839 55.918 1.00 35.82
    ATOM 1338 CB GLU 172 −3.678 24.007 56.911 1.00 37.88
    ATOM 1339 CG GLU 172 −4.713 23.810 58.011 1.00 43.69
    ATOM 1340 CD GLU 172 −6.141 23.889 57.503 1.00 47.30
    ATOM 1341 OE1 GLU 172 −6.339 23.631 56.293 1.00 49.76
    ATOM 1342 OE2 GLU 172 −7.058 24.193 58.312 1.00 47.70
    ATOM 1343 C GLU 172 −2.494 23.092 54.895 1.00 36.71
    ATOM 1344 O GLU 172 −2.769 23.503 53.756 1.00 38.19
    ATOM 1345 N ALA 173 −1.241 22.839 55.284 1.00 37.14
    ATOM 1346 CA ALA 173 −0.103 23.053 54.367 1.00 37.45
    ATOM 1347 CB ALA 173 1.230 22.912 55.109 1.00 36.11
    ATOM 1348 C ALA 173 −0.153 22.079 53.201 1.00 36.09
    ATOM 1349 O ALA 173 0.136 22.428 52.066 1.00 39.05
    ATOM 1350 N ALA 174 −0.513 20.843 53.472 1.00 35.66
    ATOM 1351 CA ALA 174 −0.598 19.905 52.375 1.00 36.41
    ATOM 1352 CB ALA 174 −0.873 18.518 52.901 1.00 36.97
    ATOM 1353 C ALA 174 −1.715 20.363 51.419 1.00 33.92
    ATOM 1354 O ALA 174 −1.558 20.279 50.213 1.00 33.46
    ATOM 1355 N LEU 175 −2.827 20.862 51.970 1.00 34.46
    ATOM 1356 CA LEU 175 −3.958 21.331 51.162 1.00 33.61
    ATOM 1357 CB LEU 175 −5.154 21.716 52.058 1.00 34.33
    ATOM 1358 CG LEU 175 −6.356 22.318 51.296 1.00 32.56
    ATOM 1359 CD1 LEU 175 −6.771 21.397 50.123 1.00 28.31
    ATOM 1360 CD2 LEU 175 −7.512 22.540 52.259 1.00 29.36
    ATOM 1361 C LEU 175 −3.612 22.518 50.263 1.00 33.20
    ATOM 1362 O LEU 175 −3.888 22.503 49.064 1.00 31.43
    ATOM 1363 N ALA 176 −3.012 23.542 50.863 1.00 34.82
    ATOM 1364 CA ALA 176 −2.616 24.746 50.146 1.00 34.42
    ATOM 1365 CB ALA 176 −1.953 25.712 51.106 1.00 35.13
    ATOM 1366 C ALA 176 −1.668 24.418 48.989 1.00 37.83
    ATOM 1367 O ALA 176 −1.868 24.882 47.866 1.00 38.78
    ATOM 1368 N ASP 177 −0.630 23.627 49.248 1.00 39.20
    ATOM 1369 CA ASP 177 0.281 23.279 48.171 1.00 41.20
    ATOM 1370 CB ASP 177 1.397 22.365 48.664 1.00 45.40
    ATOM 1371 CG ASP 177 2.318 23.065 49.640 1.00 53.11
    ATOM 1372 OD1 ASP 177 2.689 24.232 49.361 1.00 54.79
    ATOM 1373 OD2 ASP 177 2.687 22.457 50.674 1.00 55.87
    ATOM 1374 C ASP 177 −0.473 22.597 47.047 1.00 41.24
    ATOM 1375 O ASP 177 −0.196 22.834 45.872 1.00 39.86
    ATOM 1376 N LYS 178 −1.433 21.749 47.408 1.00 41.20
    ATOM 1377 CA LYS 178 −2.226 21.049 46.402 1.00 42.30
    ATOM 1378 CB LYS 178 −3.077 19.959 47.065 1.00 44.35
    ATOM 1379 CG LYS 178 −3.957 19.180 46.096 1.00 44.13
    ATOM 1380 CD LYS 178 −3.153 18.478 45.013 1.00 41.10
    ATOM 1381 CE LYS 178 −2.255 17.422 45.586 1.00 39.67
    ATOM 1382 NZ LYS 178 −1.734 16.546 44.503 1.00 41.20
    ATOM 1383 C LYS 178 −3.105 22.011 45.584 1.00 41.77
    ATOM 1384 O LYS 178 −3.145 21.916 44.365 1.00 43.66
    ATOM 1385 N GLU 179 −3.802 22.942 46.227 1.00 41.77
    ATOM 1386 CA GLU 179 −4.601 23.892 45.453 1.00 43.66
    ATOM 1387 CB GLU 179 −5.490 24.756 46.351 1.00 43.35
    ATOM 1388 CG GLU 179 −6.538 23.967 47.098 1.00 47.58
    ATOM 1389 CD GLU 179 −7.658 24.835 47.654 1.00 49.87
    ATOM 1390 OE1 GLU 179 −8.292 25.571 46.855 1.00 51.89
    ATOM 1391 OE2 GLU 179 −7.916 24.775 48.880 1.00 48.60
    ATOM 1392 C GLU 179 −3.685 24.797 44.629 1.00 44.17
    ATOM 1393 O GLU 179 −4.041 25.192 43.526 1.00 46.42
    ATOM 1394 N ALA 180 −2.511 25.132 45.163 1.00 44.61
    ATOM 1395 CA ALA 180 −1.559 25.979 44.438 1.00 45.76
    ATOM 1396 CB ALA 180 −0.359 26.289 45.300 1.00 43.35
    ATOM 1397 C ALA 180 −1.129 25.217 43.197 1.00 47.11
    ATOM 1398 O ALA 180 −0.914 25.792 42.133 1.00 48.59
    ATOM 1399 N GLU 181 −1.028 23.903 43.352 1.00 47.73
    ATOM 1400 CA GLU 181 −0.651 23.002 42.277 1.00 48.37
    ATOM 1401 CB GLU 181 −0.452 21.600 42.874 1.00 50.12
    ATOM 1402 CG GLU 181 0.477 20.663 42.114 1.00 50.74
    ATOM 1403 CD GLU 181 0.533 19.276 42.751 1.00 51.08
    ATOM 1404 OE1 GLU 181 −0.476 18.535 42.693 1.00 50.45
    ATOM 1405 OE2 GLU 181 1.584 18.933 43.324 1.00 49.78
    ATOM 1406 C GLU 181 −1.792 23.000 41.238 1.00 49.22
    ATOM 1407 O GLU 181 −1.559 23.210 40.058 1.00 48.41
    ATOM 1408 N LEU 182 −3.021 22.754 41.696 1.00 50.42
    ATOM 1409 CA LEU 182 −4.208 22.735 40.832 1.00 51.74
    ATOM 1410 CB LEU 182 −5.466 22.494 41.651 1.00 49.82
    ATOM 1411 CG LEU 182 −5.941 21.106 42.089 1.00 50.S6
    ATOM 1412 CD1 LEU 182 −6.212 20.264 40.85S 1.00 49.40
    ATOM 1413 CD2 LEU 182 −4.901 20.464 42.954 1.00 48.77
    ATOM 1414 C LEU 182 −4.411 24.036 40.072 1.00 54.20
    ATOM 1415 O LEU 182 −4.818 24.041 38.910 1.00 53.28
    ATOM 1416 N MET 183 −4.149 25.134 40.768 1.00 56.78
    ATOM 1417 CA MET 183 −4.308 26.475 40.245 1.00 60.66
    ATOM 1418 CB MET 183 −4.554 27.413 41.418 1.00 61.27
    ATOM 1419 CG MET 183 −4.745 28.857 41.067 1.00 63.93
    ATOM 1420 SD MET 183 −4.901 29.814 42.586 1.00 68.53
    ATOM 1421 CE MET 183 −6.330 28.989 43.348 1.00 67.90
    ATOM 1422 C MET 183 −3.071 26.916 39.482 1.00 64.03
    ATOM 1423 O MET 183 −3.052 27.987 38.869 1.00 64.46
    ATOM 1424 N GLN 184 −2.042 26.076 39.514 1.00 67.44
    ATOM 1425 CA OLN 184 −0.777 26.383 38.860 1.00 71.23
    ATOM 1426 CB GLN 184 0.222 25.242 39.116 1.00 73.44
    ATOM 1427 CG GLN 184 1.697 25.659 39.291 1.00 76.77
    ATOM 1428 CD GLN 184 2.348 26.207 38.020 1.00 79.02
    ATOM 1429 OE1 GLN 184 1.893 27.204 37.442 1.00 79.20
    ATOM 1430 NE2 GLN 184 3.428 25.554 37.586 1.00 79.55
    ATOM 1431 C GLN 184 −0.985 26.598 37.360 1.00 72.64
    ATOM 1432 O GLN 184 −0.971 27.737 36.873 1.00 72.31
    ATOM 1433 N ALA 185 −1.189 25.507 36.633 1.00 74.64
    ATOM 1434 CA ALA 185 −1.387 25.593 35.195 1.00 76.72
    ATOM 1435 CB ALA 185 −0.351 24.721 34.473 1.00 76.74
    ATOM 1436 C ALA 185 −2.800 25.169 34.812 1.00 77.63
    ATOM 1437 O ALA 185 −3.510 25.901 34.116 1.00 79.25
    ATOM 1438 O1 HOH 302 5.786 24.929 33.706 1.00 46.39
    ATOM 1439 O1 HOH 303 16.711 7.421 13.716 1.00 40.03
    ATOM 1440 O1 HOH 304 −0.640 6.780 57.814 1.00 49.80
    ATOM 1441 O1 HOH 305 −3.664 8.728 22.988 1.00 37.04
    ATOM 1442 O1 HOH 307 −6.985 9.376 71.599 1.00 44.76
    ATOM 1443 O1 HOH 309 2.439 23.127 69.818 1.00 64.96
    ATOM 1444 O1 HOH 310 −12.357 22.793 59.997 1.00 49.37
    ATOM 1445 O1 HOH 311 −5.000 9.881 15.800 1.00 39.08
    ATOM 1446 O1 HOH 312 −1.379 2.409 21.615 1.00 37.20
    ATOM 1447 O1 HOH 314 4.226 11.609 56.051 1.00 61.48
    ATOM 1448 O1 HOH 315 3.062 22.032 74.337 1.00 57.32
    ATOM 1449 O1 HOH 316 2.291 19.201 55.609 1.00 32.55
    ATOM 1450 O1 HOH 317 −14.260 15.606 65.187 1.00 48.61
    ATOM 1451 O1 HOH 318 1.372 26.408 49.218 1.00 45.06
    ATOM 1452 O1 HOH 320 −6.791 23.072 66.093 1.00 55.37
    ATOM 1453 O1 HOH 321 −5.960 5.653 7.525 1.00 57.42
    ATOM 1454 O1 HOH 322 9.023 7.824 4.729 1.00 49.88
    ATOM 1455 O1 HOH 323 −5.164 25.011 53.787 1.00 48.29
    ATOM 1456 O1 HOH 324 −7.620 15.194 35.505 1.00 42.96
    ATOM 1457 O1 HOH 325 0.761 16.491 65.949 1.00 49.53
    ATOM 1458 O1 HOH 327 −13.358 14.152 48.865 1.00 49.48
    ATOM 1459 O1 HOH 328 9.574 23.698 19.099 1.00 70.52
    ATOM 1460 O1 HOH 329 −15.006 12.044 46.069 1.00 61.30
    ATOM 1461 O1 HOH 330 14.950 11.135 27.309 1.00 82.65
    ATOM 1462 O1 HOH 331 −6.107 10.675 18.376 1.00 43.27
    ATOM 1463 O1 HOH 332 −17.369 21.115 39.828 1.00 59.43
    ATOM 1464 O1 HOH 333 3.753 16.179 57.494 1.00 71.35
    ATOM 1465 O1 HOH 334 −15.680 10.875 74.742 1.00 61.46
    ATOM 1466 O1 HOH 335 −15.080 23.421 48.843 1.00 55.54
    ATOM 1467 O1 HOH 336 −17.698 17.552 36.236 1.00 49.63
    ATOM 1468 O1 HOH 337 −3.687 11.077 13.706 1.00 31.62
    ATOM 1469 O1 HOH 338 3.927 6.239 5.123 1.00 44.26
    ATOM 1470 O1 HOH 339 2.369 24.751 52.035 1.00 50.57
    ATOM 1471 O1 HOH 340 −15.269 15.498 54.910 1.00 75.04
    ATOM 1472 O1 HOH 341 15.978 10.746 6.920 1.00 59.72
    ATOM 1473 O1 HOH 342 −4.143 18.035 17.666 1.00 41.15
    ATOM 1474 O1 HOH 343 −14.940 13.136 37.292 1.00 58.44
    ATOM 1475 O1 HOH 344 2.563 12.714 60.213 1.00 57.07
    ATOM 1476 O1 HOH 346 1.596 13.582 86.916 1.00 70.91
    ATOM 1477 O1 HOH 347 7.838 16.595 73.384 1.00 59.18
    ATOM 1478 O1 HOH 348 −6.147 9.577 11.401 1.00 57.10
    ATOM 1479 O1 HOH 349 −14.722 27.444 46.158 1.00 90.16
    ATOM 1480 O1 HOH 350 −4.140 13.336 31.273 1.00 33.25
    ATOM 1481 O1 HOH 351 −2.060 25.041 85.618 1.00 80.45
    ATOM 1482 O1 HOH 352 −11.312 11.026 39.981 1.00 45.94
    ATOM 1483 O1 HOH 353 −17.003 20.181 54.859 1.00 71.86
    ATOM 1484 O1 HOH 354 −0.916 3.322 71.864 1.00 49.68
    ATOM 1485 O1 HOH 355 −7.005 20.501 13.831 1.00 53.66
    ATOM 1486 O1 HOH 356 −14.600 23.546 55.631 1.00 48.35
    ATOM 1487 O1 HOH 357 −17.819 23.151 50.976 1.00 52.11
    ATOM 1488 O1 HOH 358 2.254 26.577 21.628 1.00 60.67
    ATOM 1489 O1 HOH 359 0.803 16.354 50.390 1.00 52.48
    ATOM 1490 O1 HOH 360 17.200 10.484 22.120 1.00 51.01
    ATOM 1491 O1 HOH 361 −15.864 16.596 42.676 1.00 56.63
    ATOM 1492 O1 HOH 362 2.181 8.776 24.952 1.00 38.73
    ATOM 1493 O1 HOH 363 −7.968 23.240 28.701 1.00 50.39
    ATOM 1494 O1 HOH 364 −4.004 0.134 10.731 1.00 90.56
    ATOM 1495 O1 HOH 365 −3.704 10.675 31.678 1.00 30.70
    ATOM 1496 O1 HOH 366 −13.266 25.262 52.064 1.00 62.28
    ATOM 1497 O1 HOH 367 −16.916 18.510 59.343 1.00 47.69
    ATOM 1498 O1 HOH 368 −3.703 25.280 60.980 1.00 53.12
    ATOM 1499 O1 HOH 369 −0.275 16.417 37.735 1.00 66.01
    ATOM 1500 O1 HOH 370 −7.122 20.168 74.323 1.00 40.70
    ATOM 1501 O1 HOH 371 4.079 24.812 54.526 1.00 57.35
    ATOM 1502 O1 HOH 373 −9.613 19.800 80.552 1.00 64.39
    ATOM 1503 O1 HOH 374 −9.716 25.634 31.190 1.00 50.17
    ATOM 1504 O1 HOH 375 −0.532 22.524 36.466 1.00 61.07
    ATOM 1505 O1 HOH 376 −14.409 13.165 83.189 1.00 63.05
    ATOM 1506 O1 HOH 377 −11.018 21.644 53.328 1.00 44.58
    ATOM 1507 O1 HOH 378 5.370 26.188 22.596 1.00 59.67
    ATOM 1508 O1 HOH 380 −13.995 17.778 73.426 1.00 79.09
    ATOM 1509 O1 HOH 381 4.885 0.329 8.120 1.00 74.85
    ATOM 1510 O1 HOH 382 1.331 11.313 6.097 1.00 47.98
    ATOM 1511 O1 HOH 383 11.281 12.141 6.394 1.00 56.27
    ATOM 1512 O1 HOH 400 −13.284 11.946 58.880 1.00 47.79
    ATOM 1513 O1 HOH 402 −15.563 20.103 49.349 1.00 51.23
    ATOM 1514 O1 HOH 403 2.253 19.239 4.410 1.00 73.44
    ATOM 1515 O1 HOH 405 11.304 2.402 13.475 1.00 84.55
    ATOM 1516 O1 HOH 406 −0.935 13.768 34.803 1.00 48.34
    ATOM 1517 O1 HOH 407 −3.810 21.771 34.390 1.00 43.81
    ATOM 1518 O1 HOH 408 −3.265 3.486 47.748 1.00 66.97
    ATOM 1519 O1 HOH 409 1.104 9.945 9.880 1.00 49.13
    ATOM 1520 O1 HOH 410 0.422 16.427 34.414 1.00 87.71
    ATOM 1521 O1 HOH 411 −2.673 0.494 19.885 1.00 52.04
    ATOM 1522 O1 HOH 412 −3.089 13.895 92.403 1.00 59.96
    ATOM 1523 O1 HOH 413 3.194 24.668 20.476 1.00 64.45
    ATOM 1524 O1 HOH 415 −8.097 12.619 54.223 1.00 40.52
    ATOM 1525 O1 HOH 416 2.795 7.151 22.607 1.00 54.61
    ATOM 1526 O1 HOH 417 3.751 5.165 25.893 1.00 44.77
    ATOM 1527 O1 HOH 418 1.622 9.842 42.243 1.00 56.21
    ATOM 1528 O1 HOH 419 0.421 23.884 77.749 1.00 61.98
    ATOM 1529 O1 HOH 420 14.627 6.089 21.772 1.00 57.19
    ATOM 1530 O1 HOH 421 13.038 12.816 22.645 1.00 64.28
    ATOM 1531 O1 HOH 422 −11.444 26.483 43.160 1.00 58.77
    ATOM 1532 O1 HOH 423 4.279 12.562 88.202 1.00 62.72
    ATOM 1533 O1 HOH 424 −12.414 14.580 87.763 1.00 89.26
    ATOM 1534 O1 HOH 425 −16.766 14.530 45.869 1.00 96.42
    ATOM 1535 O1 HOH 426 7.867 11.318 7.942 1.00 55.18
    ATOM 1536 O1 HOH 427 4.366 8.492 6.798 1.00 70.18
    ATOM 1537 O1 HOH 428 −2.106 3.432 77.575 1.00 57.89
    ATOM 1538 O1 HOH 429 18.086 17.462 19.670 1.00 70.05
    ATOM 1539 O1 HOH 430 −6.737 8.016 94.166 1.00 59.01
    ATOM 1540 O1 HOH 431 6.971 9.475 58.674 1.00 80.86
    ATOM 1541 O1 HOH 432 −1.536 19.816 31.738 1.00 49.54
    ATOM 1542 O1 HOH 433 7.698 10.437 31.906 1.00 61.73
    ATOM 1543 O1 HOH 434 0.207 20.512 7.972 1.00 74.94
    ATOM 1544 O1 HOH 435 3.776 13.746 6.429 1.00 75.18
    ATOM 1545 O1 HOH 436 −18.571 19.732 52.567 1.00 63.53
    ATOM 1546 O1 HOH 437 −1.899 29.425 52.295 1.00 55.12
    ATOM 1547 O1 HOH 438 −6.880 6.890 17.770 1.00 49.51
    ATOM 1548 O1 HOH 439 −6.912 14.453 37.516 1.00 51.30
    ATOM 1549 O1 EOH 440 9.467 16.541 12.773 1.00 54.26
    ATOM 1550 O1 HOH 441 2.279 17.814 53.244 1.00 61.77
    ATOM 1551 O1 HOH 442 −2.650 0.463 13.822 1.00 46.01
    ATOM 1552 O1 HOH 443 −3.215 24.966 74.028 1.00 62.02
    ATOM 1553 O1 HOH 444 −4.683 14.010 33.817 1.00 65.38
    ATOM 1554 O1 HOH 445 −8.336 27.942 89.666 1.00 52.95
    ATOM 1555 O1 HOH 446 −4.502 12.079 81.403 1.00 90.88
    ATOM 1556 O1 HOH 447 −9.457 24.981 58.998 1.00 52.30
    ATOM 1557 O1 HOH 448 −9.756 25.777 88.998 1.00 62.22
    ATOM 1558 O1 HOH 449 0.421 18.268 48.931 1.00 46.66
    ATOM 1559 O1 HOH 450 2.933 26.992 56.334 1.00 74.71
    ATOM 1560 O1 HOH 451 0.759 26.113 64.001 1.00 59.06
    ATOM 1561 O1 HOH 452 1.084 6.151 31.450 1.00 56.68
    ATOM 1562 O1 HOH 453 5.884 4.519 29.654 1.00 56.39
    ATOM 1563 O1 HOH 454 −15.425 18.155 64.235 1.00 72.96
    ATOM 1564 O1 HOH 455 −0.171 13.254 39.710 1.00 74.60
    ATOM 1565 O1 HOH 456 14.866 20.258 26.412 1.00 46.29
    ATOM 1566 O1 HOH 457 −14.275 3.131 87.758 1.00 59.31
    ATOM 1567 O1 HOH 458 −4.332 5.479 80.377 1.00 79.39
    ATOM 1568 O1 HOH 459 −2.873 25.788 71.842 1.00 79.58
    ATOM 1569 O1 HOH 460 −18.438 26.604 40.069 1.00 61.56
    ATOM 1570 O1 HOH 461 −17.600 14.799 67.139 1.00 57.68
    ATOM 1571 O1 HOH 462 18.682 10.751 18.572 1.00 52.48
    ATOM 1572 O1 HOH 463 −1.155 16.670 9.382 1.00 52.73
    ATOM 1573 O1 HOH 464 −7.994 4.789 73.142 1.00 58.99
    ATOM 1574 O1 HOH 465 2.878 15.527 89.468 1.00 53.82
    ATOM 1575 O1 HOH 466 −14.384 15.627 85.915 1.00 59.37
    ATOM 1576 O1 HOH 467 2.898 22.812 45.093 1.00 59.10
    ATOM 1577 O1 HOH 468 −0.751 11.409 92.643 1.00 70.59
    ATOM 1578 O1 HOH 469 5.961 11.789 58.672 1.00 63.17
    ATOM 1579 O1 HOH 470 17.813 12.012 26.528 1.00 46.16
    ATOM 1580 O1 HOH 471 −12.149 25.188 31.130 1.00 55.19
    ATOM 1581 O1 HOH 472 −15.854 29.561 37.766 1.00 78.50
    ATOM 1582 O1 HOH 473 0.237 23.917 15.562 1.00 69.78
    ATOM 1583 O1 HOH 474 1.289 4.512 6.743 1.00 81.85
    ATOM 1584 O1 HOH 476 −8.588 25.220 33.408 1.00 70.20
    ATOM 1585 O1 HOH 477 6.612 5.903 2.424 1.00 69.70
    ATOM 1586 O1 HOH 478 −11.595 11.694 52.934 1.00 67.26
    ATOM 1587 O1 HOH 479 −6.127 9.039 42.656 1.00 74.38
    ATOM 1588 O1 HOH 480 4.050 28.730 37.477 1.00 74.96
    ATOM 1589 O1 HOH 481 8.643 22.280 26.068 1.00 66.23
    ATOM 1590 O1 HOH 482 5.057 18.842 63.792 1.00 84.51
    ATOM 1591 O1 HOH 483 11.232 20.655 13.298 1.00 78.39
    ATOM 1592 O1 HOH 484 8.429 7.903 29.673 1.00 63.19
    ATOM 1593 O1 HOH 485 2.108 19.893 73.346 1.00 55.11
    ATOM 1594 O1 HOH 486 6.372 25.790 68.263 1.00 65.80
    ATOM 1595 O1 HOH 487 −13.205 18.613 68.124 1.00 86.13
    ATOM 1596 O1 HOH 488 −8.286 11.850 17.825 1.00 45.69
    ATOM 1597 O1 HOH 489 7.059 5.906 5.738 1.00 58.55
    ATOM 1598 O1 HOH 490 2.959 27.083 27.258 1.00 52.49
    ATOM 1599 O1 HOH 491 2.930 19.627 39.466 1.00 56.92
    ATOM 1600 O1 HOH 492 13.904 13.945 10.116 1.00 53.45
    ATOM 1601 O1 HOH 493 6.999 6.052 26.735 1.00 66.18
    ATOM 1602 O1 HOH 494 18.877 11.256 9.403 1.00 62.85
    ATOM 1603 O1 HOH 495 −14.299 15.165 36.983 1.00 54.65
    ATOM 1604 O1 HOH 496 −2.145 20.024 90.307 1.00 58.77
    ATOM 1605 O1 HOH 497 12.960 14.184 7.677 1.00 44.90
    ATOM 1606 O1 HOH 498 −0.577 22.082 22.522 1.00 84.17
    ATOM 1607 O1 HOH 499 −8.915 23.163 54.891 1.00 47.87
    ATOM 1608 O1 HOH 500 −5.904 19.990 16.699 1.00 68.72
    ATOM 1609 O1 HOH 501 7.604 22.425 14.079 1.00 83.85
    ATOM 1610 O1 HOH 502 −5.745 23.331 22.836 1.00 71.67
    ATOM 1611 O1 HOH 503 12.713 6.777 5.150 1.00 68.39
    ATOM 1612 O1 HOH 504 −8.006 21.972 84.763 1.00 67.50
    ATOM 1613 O1 HOH 505 −6.453 17.333 12.456 1.00 75.63
    ATOM 1614 O1 HOH 506 −3.191 6.789 49.810 1.00 63.94
    ATOM 1615 O1 HOH 507 4.446 24.626 63.212 1.00 85.62
    ATOM 1616 O1 HOH 508 10.825 10.927 25.007 1.00 52.28
    ATOM 1617 O1 HOH 509 1.751 28.971 44.260 1.00 88.85
    ATOM 1618 O1 HOH 510 5.304 11.175 65.843 1.00 86.55
    ATOM 1619 O1 HOH 511 3.016 25.072 34.499 1.00 56.55
    ATOM 1620 O1 HOH 512 −7.777 21.602 36.643 1.00 88.35
    ATOM 1621 O1 HOH 513 11.033 20.553 20.646 1.00 42.22
    ATOM 1622 O1 HOH 514 1.225 4.220 72.811 1.00 68.80
    ATOM 1623 O1 HOH 600 −6.830 12.211 91.855 1.00 60.53
    ATOM 1625 01 HOH 602 −6.789 6.449 80.935 1.00 50.26
    ATOM 1626 O1 HOH 603 −2.982 21.809 7.218 1.00 69.39
    ATOM 1627 O1 HOH 604 13.293 8.016 2.296 1.00 61.13
    ATOM 1628 O1 HOH 605 3.299 11.650 51.045 1.00 78.04
    ATOM 1629 O1 HOH 606 −16.971 17.233 50.226 1.00 68.89
    ATOM 1630 O1 HOH 607 2.846 12.738 39.593 1.00 71.60
    ATOM 1631 O1 HOH 608 −3.147 −2.544 9.531 1.00 58.55
    ATOM 1632 O1 HOH 609 −4.861 3.305 80.691 1.00 60.83
    ATOM 1633 O1 HOH 610 6.834 13.354 71.357 1.00 64.45
    ATOM 1634 O1 HOH 611 2.963 4.487 27.971 1.00 48.70
    ATOM 1635 O1 HOH 612 −2.162 14.623 70.194 1.00 72.85
    ATOM 1636 O1 HOH 613 −8.983 11.246 91.235 1.00 68.16
    ATOM 1637 O1 HOH 614 −1.414 15.727 89.255 1.00 63.90
    ATOM 1638 O1 HOH 615 1.353 25.378 70.806 1.00 68.29
    ATOM 1639 O1 HOH 616 0.315 14.016 78.504 1.00 59.91
    ATOM 1640 O1 HOH 617 4.477 9.852 70.092 1.00 79.40
    ATOM 1641 O1 HOH 618 −12.259 11.420 46.776 1.00 58.20
    ATOM 1642 O1 HOH 619 2.679 4.422 32.337 1.00 49.91
    ATOM 1643 O1 HOH 620 −17.285 12.212 71.786 1.00 68.80
    ATOM 1644 O1 HOH 621 −17.043 8.933 85.930 1.00 71.94
    ATOM 1645 O1 HOH 622 −12.833 21.451 57.231 1.00 69.93
    ATOM 1646 O1 HOH 623 −16.934 18.199 72.267 1.00 64.58
    ATOM 1647 O1 HOH 624 −1.857 26.990 67.917 1.00 52.07
    ATOM 1648 O1 HOH 625 −13.989 23.289 43.768 1.00 68.81
    ATOM 1649 O1 HOH 626 −8.279 10.487 46.008 1.00 64.05
    ATOM 1650 O1 HOH 627 −8.410 14.523 46.407 1.00 94.70
    ATOM 1651 01 HOH 628 −14.238 29.716 34.051 1.00 74.01
    ATOM 1652 O1 HOH 629 2.497 10.302 52.957 1.00 78.36
    ATOM 1653 O1 HOH 630 12.781 18.085 14.371 1.00 59.38
    ATOM 1654 O1 HOH 631 −13.379 8.527 74.408 1.00 72.72
    ATOM 1655 O1 HOH 632 −8.436 24.292 72.901 1.00 80.80
    ATOM 1656 O1 HOH 633 −11.927 12.370 43.399 1.00 72.78
    ATOM 1657 O1 HOH 634 3.105 21.424 61.107 1.00 80.29
    ATOM 1658 O1 HOH 635 1.552 18.331 85.585 1.00 75.99
    ATOM 1659 O1 HOH 636 −13.619 20.355 61.490 1.00 63.28
    ATOM 1660 O1 HOH 637 −2.200 8.533 10.367 1.00 63.23
    ATOM 1661 O1 HOH 638 3.589 19.637 44.461 1.00 47.03
    ATOM 1662 O1 HOH 639 −1.573 21.818 9.438 1.00 68.93
    ATOM 1663 O1 HOH 640 6.258 22.557 73.322 1.00 58.13
    ATOM 1664 O1 HOH 641 −13.066 10.161 44.534 1.00 63.83
    ATOM 1665 O1 HOH 642 −9.747 28.802 92.952 1.00 80.11
    ATOM 1666 O1 HOH 643 −5.127 4.127 4.706 1.00 79.47
    ATOM 1667 O1 HOH 644 −13.918 12.183 81.003 1.00 58.64
    ATOM 1668 O1 HOH 645 19.938 9.499 13.944 1.00 87.18
    ATOM 1669 HG MER 701 9.336 0.982 15.636 0.20 58.21
    ATOM 1670 HG MER 702 9.841 5.199 14.508 0.20 67.41
    ATOM 1671 HG MER 703 −7.127 10.772 57.593 0.20 79.35
    ATOM 1672 C1 DMA 801 −13.170 18.614 25.453 1.00 77.87
    ATOM 1674 C1′ DMA 801 −11.027 17.896 26.233 1.00 77.70
    ATOM 1675 C2′ DMA 801 −9.669 18.447 26.518 1.00 75.99
    ATOM 1676 C3′ DMA 801 −8.688 17.714 25.612 1.00 72.89
    ATOM 1677 C4′ DMA 801 −7.247 18.219 25.835 1.00 69.58
    ATOM 1678 C5′ DMA 801 −6.263 17.478 24.914 1.00 65.32
    ATOM 1679 C6′ DMA 801 −6.218 16.101 25.451 1.00 61.15
    ATOM 1680 C7′ DMA 801 −5.365 15.188 24.661 1.00 59.83
    ATOM 1681 C8′ DMA 801 −5.575 13.886 25.325 1.00 64.73
    ATOM 1682 C9′ DMA 801 −4.915 12.693 24.717 1.00 64.11
    ATOM 1683 C10′ DMA 801 −5.345 11.526 25.553 1.00 67.12
    ATOM 1684 O1 DMA 801 −11.982 18.934 26.182 1.00 79.61
    ATOM 1685 O5 DMA 801 −12.859 18.362 24.066 1.00 75.75
  • While the present invention has been described with respect to certain preferred embodiments, other modifications and variations may be made without departing from the spirit and scope of the present invention as set forth in the following claims. [0098]

Claims (21)

What is claimed is:
1. A crystal of ribosome recycling factor (RRF) or an RRF homolog comprising:
RRF or an RRF mutant or an RRF homolog, which has substantially the same structure as that of RRF and whose amino acid sequence is formed by substituting, or eliminating at least one amino acid residue in the RRF amino acid sequence or by adding at least one amino acid residue to the RRF sequence; and
a compound, which can bind to the hydrophobic cleft formed between two domains of RRF.
2. The crystal of RRF or an RRF homolog according to claim 1, wherein the compound is a compound having 3 to 12 carbon atoms on a straight chain alkyl group, or a compound having 3 to 12 carbon atoms on a straight chain alkyl group and a functional group which can form a non-hydrophobic bond.
3. The crystal of RRF or an RRF homolog according to claim 1, wherein the compound is decyl-□-D-maltopyranoside.
4. The crystal of RRF or an RRF homolog according to claim 1, wherein the RRF or RRF homolog has the three-dimensional structure specified by 0.2 nm of root mean square deviation (rmsd) between the coordinate of each atom on the RRF or RRF homolog and the corresponding coordinate of each atom described in table 2.
5. A crystallization method for RRF or an RRF homolog comprising the step of crystallizing RRF, an RRF mutant or an RRF homolog with a compound binding to the hydrophobic cleft formed between two domains of RRF, wherein the RRF mutant or RRF homolog has substantially the same structure as that of RRF and wherein the amino acid sequence of the RRF mutant or homolog is formed by substituting, or eliminating at least one amino acid residue in the RRF amino acid sequence or by adding at least one amino acid residue to the RRF sequence.
6. The crystallization method according to claim 5, wherein said compound comprises 3-12 carbon atoms on a straight chain alkyl group, or 3-12 carbon atoms on a straight chain alkyl group and a functional group which can form a non-hydrophobic bond.
7. The crystallization method according to claim 5, wherein said compound is decyl-β-D-maltopyranoside.
8. An inhibitor of RRF comprising the coiled coil domain of RRF or an RRF homolog wherein said coiled coil domain binds to the hydrophobic cleft of the RRF or RRF homolog.
9. A method for identifying an inhibitor of RRF comprising the steps of:
selecting or designing an inhibitor candidate compound or its fragment from a database using the three-dimensional structure of RRF defined by the coordinates of atoms in RRF described in table 2;
inserting the compound or fragment into the three-dimensional structure of RRF using a computer modeling method;
determining the fit of the inhibitor candidate with RRF; and
selecting as an inhibitor the compound or fragment which fits in such a way that it will inhibit the activity of RRF.
10. The method according to claim 9 further comprising the steps of modifying the compound or fragment using the three-dimensional structure of RRF and repeating the steps of inserting, determining and selecting as an inhibitor.
11. The method according to claim 9 further comprising the steps of designing a compound by linking two or more of the compounds or fragments wherein said compounds or fragments bind to different parts of RRF, and repeating the steps of inserting, determining and selecting as an inhibitor.
12. The method according to claim 9 further comprising the steps of:
preparing the inhibitor of RRF;
identifying whether the compound inhibits the activity of RRF in a prokaryote in vivo or in vitro; and
modifying the inhibitor of RRF using the three-dimensional structure of RRF, and repeating the steps of inserting, determining and selecting as an inhibitor.
13. The method according to claim 9, wherein the three-dimensional structure of RRF used in the analysis is the three-dimensional structure formed by the hydrophobic cleft and its peripheral or circumferential amino acid residues.
14. The method according to claim 13, wherein the inhibitor comprises an alkyl group which binds to the hydrophobic cleft of RRF and further comprising a functional group non-hydrophobically binding to the peripheral or circumferential residues near the hydrophobic cleft.
15. The method according to claim 9, wherein the step of determining the fit of the inhibitor with RRF comprises: determining the fit of the inhibitor with RRF when the dissociation constant of the interaction is 100 micromoles or less.
16. The method according to claim 9, wherein the three dimensional structure of RRF used for the analysis is that of the coiled coil domain of RRF.
17. The method according to claim 16 further comprising the steps of:
preparing an RRF mutant, in which at least one residue is substituted on the surface of the coiled coil domain of RRF;
preparing a prokaryote which expresses the RRF mutant; and
identifying whether the RRF mutant reduces the growth of said prokaryote
designing a compound comprising at least the substituted residue in the RRF;
preparing the compound;
identifying the inhibition activity of the compound.
18. An isolated polypeptide comprising substantially the same three-dimensional structure as that of the coiled coil domain of RRF.
19. The isolated polypeptide of claim 18 comprising sequences selected from the group consisting of SEQ ID NOS:1-4 or truncated versions thereof.
20. A method for identifying an inhibitor of RRF comprising the steps of:
preparing a mutant, in which at least one residue is substituted on the surface of the coiled coil domain of RRF;
checking the growth of a prokaryote expressing the mutant RRF;
identifying those mutants of RRF that cause said prokaryotes to have a reduced growth rate as containing mutation at residues involved in binding of RRF to the ribosome.
21. The method according to claim 20, wherein when the residues in the binding site or part are isolated by other residue(s) in the domain, the compound is designed by linking the residues in the binding site or part with linker in the designing step.
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